TFPI inhibitors and methods of use

ABSTRACT

The invention provides peptides that bind Tissue Factor Pathway Inhibitor (TFPI), including TFPI-inhibitory peptides, and compositions thereof. Peptide complexes also are provided. The peptides may be used to inhibit a TFPI, enhance thrombin formation in a clotting factor-deficient subject, increase blood clot formation in a subject, treat a blood coagulation disorder in a subject, purify TFPI, and identify a TFPI-binding compound.

CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 61/613,865, filed Mar. 21, 2012, which is herebyincorporated by reference in its entirety. The following applicationsalso are incorporated by reference in their entirety: U.S. ProvisionalPatent Application No. 61/139,272, filed Dec. 19, 2008; U.S. patentapplication Ser. No. 12/643,818, filed Dec. 21, 2009; InternationalPatent Application No. PCT/US2009/069060, filed Dec. 21, 2009; U.S.Provisional Patent Application No. 61/315,758, filed Mar. 19, 2010; U.S.patent application Ser. No. 13/026,070, filed Feb. 11, 2011; andInternational Patent Application No. PCT/US2011/024604, filed Feb. 11,2011.

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: ASCII text file named“44241E_SeqListing.txt,” 1,248,600 bytes, created Jan. 31, 2013.

TECHNICAL FIELD OF THE INVENTION

The invention generally relates to peptides that bind Tissue FactorPathway Inhibitor (TFPI) and uses thereof.

BACKGROUND OF THE INVENTION

Hemostasis relies on the complex coagulation cascade, wherein a seriesof events mediated by blood clotting factors leads to conversion ofprothrombin to thrombin. Factor X (FX) activation is the central eventof both the intrinsic and extrinsic pathways of the coagulation cascade.The extrinsic pathway has been proposed as the primary activator of thecoagulation cascade (Mackman et al., Arterioscler. Thromb. Case. Biol.,27, 1687-1693 (2007)). Circulating Tissue Factor (TF) and activatedFactor VII (FVIIa) interact to form the “extrinsic complex,” whichmediates activation of FX. The coagulation cascade is amplified by theintrinsic pathway, during which successive activation of factors XII,XI, IX, and VIII results in formation of the “intrinsic” FIXa-FVIIIacomplex that also mediates FX activation. Activated FX promotes thrombinformation, which is required for the body to create fibrin andeffectively curb bleeding.

Severe bleeding disorders, such as hemophilia, result from disruption ofthe blood coagulation cascade. Hemophilia A, the most common type ofhemophilia, stems from a deficiency in factor VIII, while hemophilia Bis associated with deficiencies in Factor IX (FIX). Hemophilia C iscaused by a deficiency in Factor XI (FXI) (Cawthern et al., Blood,91(12), 4581-4592 (1998)). There is currently no cure for hemophilia andother clotting diseases. Factor replacement therapy is the most commontreatment for blood coagulation disorders. However, blood clottingfactors typically are cleared from the bloodstream shortly afteradministration. To be effective, a patient must receive frequentintravenous infusions of plasma-derived or recombinant factorconcentrates, which is uncomfortable, requires clinical settings, isexpensive, and is time consuming. In addition, therapeutic efficacy offactor replacement therapy can diminish drastically upon formation ofinhibitory antibodies. Approximately 30% of patients with severehemophilia A develop inhibitory antibodies that neutralize Factor VIII(FVIII) (Peerlinck and Hermans, Haemophilia, 12, 579-590 (2006)). Fewtherapeutic options exist for patients with anti-Factor antibodies.

Thus, there exists a need in the art for compositions and methods fortreating blood coagulation disorders. The invention provides suchcompositions and methods.

SUMMARY OF THE INVENTION

The invention includes, for example, a peptide complex comprising afirst peptide and a second peptide, wherein the peptide complexcomprises 30-60 amino acids. In various embodiments, two or morepeptides as described herein (e.g., peptides encompassed by any offormulas (I) to (XIV)) are linked or fused to create a peptide complex(e.g., a dimer comprising two peptides (which may be the same ordifferent), a trimer comprising three peptides (two or more of which maybe the same or different), etc.). In various embodiments, the peptidecomplex binds to at least two different TFPI epitopes and, optionally,inhibits at least two functions of TFPI. In some embodiments, thepeptide or peptide complex of the invention binds TFPI-1 (e.g., TFPI-1α)and, optionally, improves TFPI-regulated thrombin generation in theabsence of FVIII, FIX, and/or FXI. A composition (e.g., a pharmaceuticalcomposition) comprising the peptide complex also is provided.

In addition, the invention provides methods of using the peptide orpeptide complex of the invention. For example, the invention provides amethod of inhibiting a TFPI comprising contacting the TFPI with apeptide as described herein. The invention also provides a method ofenhancing thrombin formation in a clotting factor-deficient subject, amethod for increasing blood clot formation in a subject, and a method oftreating a blood coagulation disorder in a subject. The methods are, intheir entirety, also referred to herein as, e.g., “the method of theinvention.” The methods comprise administering to the subject a peptideor peptide complex as provided herein in an amount effective to achievea desired effect, e.g., an amount effective to enhance thrombinformation, an amount effective to enhance blood clot formation, or anamount effective to treat the blood coagulation disorder in the subject.Further aspects of the invention include use of the peptide of theinvention for the manufacture of a medicament, a method for targeting acell displaying TFPI, a method for treating or diagnosing a subjectsuffering from a disease or at risk of suffering from a disease, amethod of purifying TFPI, and a method of identifying a TFPI-bindingcompound. Unless explicitly indicated to the contrary, the descriptionprovided herein with respect to one peptide of the invention or methodof the invention applies to each and every peptide of the invention andmethod of the invention, respectively. Also, unless explicitly indicatedto the contrary, the description provided herein with respect to apeptide of the invention or use thereof applies to peptide complexes ofthe invention.

The invention also includes a TFPI inhibitor that binds human TFPI at afirst binding site defined by amino acid residues F28, K29, A30, D32,I46, F47, and I55 and a second binding site defined by amino acidresidues R41, Y53, C59, E60, Q63, R65, E67, E71, K74, M75, N80, N82,R83, I84, I85, T87, F96, C106, C130, L131, N133, M134, N136, F137, E142,N145, and I146. A method for identifying a TFPI-binding compound also isprovided. The method comprises (a) contacting a peptide comprising TFPIKunitz domain 1 (KD1) and Kunitz domain 2 (KD2) with a test compound,and (b) detecting binding of the test compound to a TFPI binding sitedefined by KD1-KD2 amino acid residues corresponding to human TFPIresidues R41, Y53, C59, E60, Q63, R65, E67, E71, K74, M75, N80, N82,R83, I84, I85, T87, F96, C106, C130, L131, N133, M134, N136, F137, E142,N145, and I146.

The following numbered paragraphs each succinctly define one or moreexemplary variations of the invention:

1. A peptide complex comprising a first peptide and a second peptide,wherein the peptide complex comprises 30-60 amino acids and binds to atleast two different TFPI epitopes and inhibits two or more TFPIfunctions.

2. A peptide complex comprising a first peptide and a second peptide,wherein

(a) the first peptide comprises the structure of formula (XIII):

X6001-X6002-X6003-X6004-X6005-X6006-X6007-X6008-X6009-X6010-X6011-X6012-X6013-X6014-X6015-X6016-X6017-X6018-X6019-X6020(XIII) (SEQ ID NO: 3153);

wherein X6001 is an amino acid selected from the group consisting of F,L, M, Y, 1Ni, Thi, Bta, Dopa, Bhf, C, D, G, H, I, K, N, Nmf, Q, R, T, V,and W; wherein X6002 is an amino acid selected from the group consistingof Q, G, and K; wherein X6003 is an amino acid selected from the groupconsisting of C, D, E, M, Q, R, S, T, Ede(O), Cmc, A, Aib, Bhs, F, G, H,I, K, L, N, P, V, W and Y; wherein X6004 is an amino acid selected fromthe group consisting of Aib, E, G, I, K, L, M, P, R, W, Y, A, Bhk, C, D,F, H, k, N, Nmk, Q, S, T and V; wherein X6005 is an amino acid selectedfrom the group consisting of a, A, Aib, C, D, d, E, G, H, K, k, M, N,Nmg, p, Q, R, NpropylG, aze, pip, tic, oic, hyp, nma, Ncg, Abg, Apg,thz, dtc, Bal, F, L, S, T, V, W and Y; wherein X6006 is an amino acidselected from the group consisting of A, C, C(NEM), D, E, G, H, K, M, N,Q, R, S, V, Cit, C(Acm), Nle, I, Ede(O), Cmc, Ed, Eea, Eec, Eef, Nif,Eew, Aib, Btq, F, L, T, W and Y; wherein X6007 is an amino acid selectedfrom the group consisting of I, V, T, Chg, Phg, Tle, A, F, G, K, L, Nmv,P, Q, S, W and Y; wherein X6008 is an amino acid selected from the groupconsisting of F, H, 1Ni, 2Ni, Pmy, Y, and W; wherein X6009 is an aminoacid selected from the group consisting of Aib, V, Chg, Phg, Abu, Cpg,Tle, L-2-amino-4,4,4-trifluorobutyric acid, A, f, I, K, S, and T;wherein X6010 is an amino acid selected from the group consisting of A,C, D, d, E, F, H, K, M, N, P, Q, R, S, T, V, W, Y, Nmd, C(NEM), Aib, G,I, L and Nmf; wherein X6011 is an amino acid selected from the groupconsisting of A, a, G, p, Sar, c, hcy, Aib, C, K, G and Nmg; whereinX6012 is an amino acid selected from the group consisting of Y, Tym,Pty, Dopa and Pmy; wherein X6013 is an amino acid selected from thegroup consisting of Aib, C, F, 1Ni, Thi, Bta, A, E, G, H, K, L, M, Q, R,W and Y; wherein X6014 is an amino acid selected from the groupconsisting of A, Aib, C, C(NEM), D, E, K, L, M, N, Q, R, T, V, Hcy, Bhe,F, G, H, I, P, S, W and Y; wherein X6015 is an amino acid selected fromthe group consisting of R, (omega-methyl)-R, D, E and K; wherein X6016is an amino acid selected from the group consisting of L, Hcy, Hle andAml; wherein X6017 is an amino acid selected from the group consistingof A, a, Aib, C, c, Cha, Dab, Eag, Eew, H, Har, Hci, Hle, I, K, L, M,Nle, Nva, Opa, Orn, R, S, Deg, Ebc, Eca, Egz, Aic, Apc, Egt,(omega-methyl)-R, Bhr, Cit, D, Dap, E, F, G, N, Q, T, V, W and Y;wherein X6018 is an amino acid selected from the group consisting of A,Aib, Hcy, hcy, C, c, L, Nle, M, N, R, Bal, D, E, F, G, H, I, K, Q, S, T,V, W and Y; wherein X6019 is an amino acid selected from the groupconsisting of K, R, Har, Bhk and V; and wherein X6020 is an amino acidselected from the group consisting of K, L, Hcy, Aml, Aib, Bhl, C, F, G,H, I, Nml, Q, R, S, T, V, W and Y, and

(b) the second peptide comprises the structure of formula (XIV):

X7001-X7002-X7003-X7004-X7005-X7006-[X7007-X7008-X7009-X7010-X7011-X7012-X7013-X7014-X7015-X7016-X7017-X7018]-X7019-X7020-X7021-X7022-X7023(XIV) (SEQ ID NO: 3154),

wherein X7001 is either present or absent, whereby in case X7001 ispresent it is an amino acid selected from the group consisting of A, C,C(NEM), D, E, F, G, H, I, K, L, P, R, S, T, V and W; wherein X7002 iseither present or absent, whereby in case X7002 is present it is anamino acid selected from the group consisting of A, C, C(NEM), D, E, F,G, H, I, K, L, M, P, Q, R, S, T, V, W and Y; wherein X7003 is an aminoacid selected from the group consisting of A, F, I, K, L, R, S, T, V, Wand Y; wherein X7004 is an amino acid selected from the group consistingof A, D, E, F, G, I, K, L, R, S, T, V and W; wherein X7005 is R or W;wherein X7006 is an amino acid selected from the group consisting of F,H, I, K, L, R, V and W; wherein X7007 is an amino acid selected from thegroup consisting of Orn, homoK, C, Hcy, Dap and K, preferably selectedfrom the group consisting of C and Hcy; wherein X7008 is an amino acidselected from the group consisting of A, G, R, S and T; wherein X7009 isan amino acid selected from the group consisting of a, A, I, K, L, M, m,Moo, Nle, p, R, Sem and V; wherein X7010 is an amino acid selected fromthe group consisting of A, G, I, K, L, P, R, S, T and V; wherein X7011is an amino acid selected from the group consisting of D, E, G, S and T;wherein X7012 is an amino acid selected from the group consisting of A,a, D, d, E, e, F, f, G, I, K, k, L, l, M, m, Moo, Nle, nle, P, p, R, r,S, s, Sem, T, t, V, v, W and w; wherein X7013 is an amino acid selectedfrom the group consisting of A, C, C(NEM), Con, Con(Meox), D, d, E, e,Eag, F, G, I, K, L, N, R, S, s, T, V and W; wherein X7014 is an aminoacid selected from the group consisting of A, D, E, F, G, I, K, L, M, R,S, T, V and W; wherein X7015 is an amino acid selected from the groupconsisting of A, D, E, F, G, I, K, L, M, Nle, R, S, T, V and W; whereinX7016 is an amino acid selected from the group consisting of A, D, E, F,I, K, L, M, Moo, Nle, R, S, Sem, T, V, W and Y; wherein X7017 is anamino acid selected from the group consisting of A, D, E, F, G, I, K, L,R, S, T, V, W and Y; wherein X7018 is an amino acid selected from thegroup consisting of C and D, preferably C; wherein X7019 is an aminoacid selected from the group consisting of A, F, I, L, S, T, V and W;wherein X7020 is an amino acid selected from the group consisting of Fand W; wherein X7021 is an amino acid selected from the group consistingof I, L and V; wherein X7022 is an amino acid selected from the groupconsisting of A, D, E, F, G, I, K, L, P, R, S, T, V and W; wherein X7023is either present or absent, whereby in case X7023 is present it is anamino acid selected from the group consisting of A, C, C(NEM), Con,Con(Meox), D, E, Eag, F, G, I, K, L, R, S, T, V, W and Y; and whereinthe peptide comprises as a cyclic structure generated by a linkagebetween X7007 and X7018.

3. The peptide complex of paragraph 2,

wherein X6001 is an amino acid selected from the group consisting of1Ni, Bta, Dopa, F, L, Y and M; wherein X6002 is Q; wherein X6003 is anamino acid selected from the group consisting of D, E, S, M, Q, R, T andC; wherein X6004 is an amino acid selected from the group consisting ofK, Aib, L, P, R, E, G, I, Y, M and W; wherein X6005 is an amino acidselected from the group consisting of p, Nmg, NpropylG, aze, pip, tic,oic, hyp, a, Aib, D, d, G, H, K, k, N, Q, R, A, E, C and M; whereinX6006 is an amino acid selected from the group consisting of C, E, K, R,S, V, C(Acm), Nle, C(NEM), I, Cit, A, D, G, H, N, Q and M; wherein X6007is an amino acid selected from the group consisting of Tle, V and I;wherein X6008 is an amino acid selected from the group consisting of H,1Ni, 2Ni, Pmy, F and Y; wherein X6009 is V, Abu or Tle; wherein X6010 isan amino acid selected from the group consisting of D, P, C, T, A, E, K,M, N, Q, R, F, H, S, V, W and Y; wherein X6011 is G, a, c, hcy or Sar;wherein X6012 is Y; wherein X6013 is an amino acid selected from thegroup consisting of F, 1Ni, Bta and C; wherein X6014 is an amino acidselected from the group consisting of Aib, C, E, Hcy, A, D, K, L, M, N,Q, R, T, V and Aib; wherein X6015 is R; wherein X6016 is an amino acidselected from the group consisting of L, Aml, Hle and Hcy; wherein X6017is an amino acid selected from the group consisting of A, Aib, C, c,Aic, Eca, Deg, Cha, Dab, Dap, Eag, Eew, H, Har, Hci, Hle, K, Nle, Nva,Opa, Orn, R, I, L, S and M; wherein X6018 is an amino acid selected fromthe group consisting of A, Aib, C, c, L, Hcy, N, M and R; wherein X6019is K; and wherein X6020 is an amino acid selected from the groupconsisting of L, Aml, Hcy and K.

4. The peptide complex of paragraph 2 or paragraph 3, wherein the firstpeptide and/or second peptide further comprises N-terminal amino acid(s)and/or moieties linked to X6001 and/or X7001 and selected from the groupconsisting of FAM-Ttds, PE, Palm, 2-phenyl acetyl, 3-phenyl propionyl,2-(naphth-2-yl) acetyl, hexanoyl, 2-methyl propionyl, 3-methyl butanoyl,2-naphthylsulfonyl, 1-naphthylsulfonyl, acetyl, Con, Con(Meox), AOA,Oxme-AOA, Meox-Lev, levulinic acid (Lev), and pentynoic acid (Pyn).

5. The peptide complex of any one of paragraphs 2-4, wherein the firstpeptide and/or second peptide further comprises X6021 linked to X6020 orX7024 linked to X7023, respectively, wherein X6021 and/or X7024comprises C-terminal amino acid(s) and/or moieties selected from thegroup consisting of Hly, K, Orn, Dab, Eag, Dap, Hcy, Pen, C, c, C(NEM),Con, Con(Meox), K(Ttds-maleimidopropionyl(EtSH)), K(Tdts-maleimid),K(AOA), K(Myr), K(Ttds-Myr), K(Ttds-Palm), K(Ttds-Ac), K(Ttds-γGlu-Myr),K(AlbuTag), K(4PBSA), Cea, and amide.

6. The peptide complex of any one of paragraphs 2-5,

wherein X7001 is an amino acid selected from the group consisting of A,D, F, G, H, K, L and S; wherein X7002 is an amino acid selected from thegroup consisting of H, F, M and R; wherein X7003 is an amino acidselected from the group consisting of F and Y; wherein X7004 is K;wherein X7005 is W; wherein X7006 is an amino acid selected from thegroup consisting of F and H; wherein X7007 is C; wherein X7008 is anamino acid selected from the group consisting of A, G and S; whereinX7009 is an amino acid selected from the group consisting of M, Sem andV; wherein X7010 is an amino acid selected from the group consisting ofK, P and R; wherein X7011 is D; wherein X7012 is an amino acid selectedfrom the group consisting of F, L, l, M and Sem; wherein X7013 is anamino acid selected from the group consisting of D, G, K and S; whereinX7014 is G; wherein X7015 is an amino acid selected from the groupconsisting of I and T; wherein X7016 is an amino acid selected from thegroup consisting of D, F, M, Sem and Y; wherein X7017 is an amino acidselected from the group consisting of S and T; wherein X7018 is C;wherein X7019 is an amino acid selected from the group consisting of Aand V; wherein X7020 is W; wherein X7021 is V; wherein X7022 is an aminoacid selected from the group consisting of F, L, K, R, P and W; whereinX7023 is either present or absent, whereby in case X7023 is present itis an amino acid sequence selected from the group consisting of A, D, F,M, S and Y; and wherein the peptide comprises as a cyclic structuregenerated by a linkage between X7007 and X7018.

7. The peptide complex of any one of paragraphs 1-6 wherein the firstpeptide and the second peptide is linked by linker moiety, preferablyabout 1-100 Å in length.

8. The peptide complex of paragraph 7, wherein the linker moiety isabout 5-50 Å in length.

9. The peptide complex of paragraph 8, wherein the linker moiety isabout 10-30 Å in length.

10. The peptide complex of any one of paragraphs 7-9, wherein the linkermoiety comprises the structure Z₁₋₂₀, wherein Z is an amino acid,hydroxy acid, ethylene glycol, propylene glycol, or a combination of anyof the foregoing.

11. The peptide complex of paragraph 10, wherein Z is G, s, S, a, A,Bal, Gaba, Ahx, Ttds, or a combination of any of the foregoing.

12. The peptide complex of any one of paragraphs 7-11, wherein thelinker moiety is attached to the first peptide and/or the second peptidevia an oxime, a hydrazide, a succinimide, a thioether, a triazole, asecondary amine, an amide, or a disulfide.

13. The peptide complex of any one of paragraphs 7-12, wherein theC-terminus of the first peptide is linked to the N-terminus of thesecond peptide via the linker moiety.

14. The peptide complex of any one of paragraphs 7-13, wherein the firstpeptide comprises the structure of formula (XIII), and linker moietyattaches to the first peptide at the N-terminus, at the C-terminus, orat side chains of X6001, X6004, X6006, X6010, X6014, or X6020.

15. The peptide complex of any one of paragraphs 1-14, wherein the firstpeptide comprises an amino acid sequence at least 80%, at least 85%, atleast 90%, at least 95%, or 100% identical to SEQ ID NO: 178 or SEQ IDNO: 4261.

16. The peptide complex of any one of paragraphs 1-15, wherein thesecond peptide comprises an amino acid sequence at least 80%, at least85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1044.

17. The peptide complex of paragraph 1 or paragraph 2 comprising theamino acid sequence set forth in SEQ ID NO: 4260.

18. The peptide complex of any one of paragraphs 1-17, wherein thepeptide complex is conjugated to a polyethylene glycol (PEG) moiety,human serum albumin (HSA), an HSA-binding domain, an antibody orfragment thereof, hydroxyethyl starch, a proline-alanine-serine multimer(PASylation), a C₁₂-C₁₈ fatty acid, or polysialic acid.

19. A TFPI inhibitor that binds human TFPI at a first binding sitedefined by amino acid residues F28, K29, A30, D32, I46, F47, and I55 anda second binding site defined by amino acid residues R41, Y53, C59, E60,Q63, R65, E67, E71, K74, M75, N80, N82, R83, I84, I85, T87, F96, C106,C130, L131, N133, M134, N136, F137, E142, N145, and I146.

20. The TFPI inhibitor of paragraph 19, where the first binding site isdefined by amino acid residues A27, F28, K29, A30, D31, D32, K36, I38,I46, F47, and I55.

21. The TFPI inhibitor of paragraph 20, where the first binding site isdefined by amino acid residues A27, F28, K29, A30, D31, D32, K36, A37,I38, F44, I46, F47, and I55.

22. The TFPI inhibitor of any one of paragraphs 19-21 which is apeptide.

23. The TFPI inhibitor of any one of paragraphs 19-21, comprising afirst peptide and a second peptide linked by a linker moiety.

24. The TFPI inhibitor of paragraph 23, wherein the linker moiety isabout 1-100 Å in length, about 5-50 Å, or about 10-30 Å in length.

25. The TFPI inhibitor of paragraph 23 or paragraph 24, wherein thelinker moiety comprises the structure Z₁₋₂₀, wherein Z is an amino acid,hydroxy acid, ethylene glycol, propylene glycol, or a combination of anyof the foregoing.

26. The TFPI inhibitor of paragraph 25, wherein Z is G, s, S, a, A, Bal,Gaba, Ahx, Ttds, or a combination of any of the foregoing.

27. A peptide complex of any one of paragraphs 1-18 or a TFPI inhibitorof any one of paragraphs 19-26 for use in a method for the treatment ofa subject.

28. The peptide complex or TFPI inhibitor of paragraph 27, wherein themethod is for the treatment of a blood coagulation disorder.

29. Use of the peptide complex of any one of paragraphs 1-18 or the TFPIinhibitor of any one of paragraphs 19-26 for the manufacture of amedicament.

30. Use of the peptide complex of any one of paragraphs 1-18 or the TFPIinhibitor of any one of paragraphs 19-26 for the manufacture of amedicament for the treatment of a blood coagulation disorder.

31. A pharmaceutical composition comprising the peptide complex of anyone of paragraphs 1-18 or the TFPI inhibitor of any one of paragraphs19-26 and a pharmaceutically acceptable carrier.

32. The pharmaceutical composition of paragraph 31, wherein thecomposition comprises a further pharmaceutically effective agent.

33. The pharmaceutical composition of paragraph 31, wherein thepharmaceutical composition is for use in a method of treating a bloodcoagulation disorder.

34. A method for treating a subject suffering from a disease or being atrisk of suffering from a disease, the method comprising administering tothe subject a pharmaceutical composition of paragraph 31.

35. The method of paragraph 34, wherein the disease is a bloodcoagulation disorder.

36. A method for identifying a TFPI-binding compound, the methodcomprising (a) contacting a peptide comprising TFPI Kunitz domain 1(KD1) and Kunitz domain 2 (KD2) with a test compound, and (b) detectingbinding of the test compound to a TFPI binding site defined by KD1-KD2amino acid residues corresponding to human TFPI residues R41, Y53, C59,E60, Q63, R65, E67, E71, K74, M75, N80, N82, R83, I84, I85, T87, F96,C106, C130, L131, N133, M134, N136, F137, E142, N145, and I146.

37. The method of paragraph 36, wherein step (b) further comprisesdetecting binding of the test compound to a TFPI binding site defined byKD1 amino acid residues corresponding to human TFPI residues F28, K29,A30, D32, I46, F47, and I55.

38. The method of paragraph 37, wherein the TFPI binding site is definedby KD1 amino acid residues corresponding to human TFPI residues F28,K29, A30, D32, I46, F47, I55A27, D31, K36, A37, I38, F44, and 146.

39. A peptide consisting of the amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1337-1355 and 4240-4268.

40. A method for inhibiting degradation of TFPI by a serine protease,the method comprising contacting TFPI with a peptide comprising thestructure of formula (XIV):

X7001-X7002-X7003-X7004-X7005-X7006-[X7007-X7008-X7009-X7010-X7011-X7012-X7013-X7014-X7015-X7016-X7017-X7018]-X7019-X7020-X7021-X7022-X7023(XIV) (SEQ ID NO: 3154),

wherein X7001 is either present or absent, whereby in case X7001 ispresent it is an amino acid selected from the group consisting of A, C,C(NEM), D, E, F, G, H, I, K, L, P, R, S, T, V and W; wherein X7002 iseither present or absent, whereby in case X7002 is present it is anamino acid selected from the group consisting of A, C, C(NEM), D, E, F,G, H, I, K, L, M, P, Q, R, S, T, V, W and Y; wherein X7003 is an aminoacid selected from the group consisting of A, F, I, K, L, R, S, T, V, Wand Y; wherein X7004 is an amino acid selected from the group consistingof A, D, E, F, G, I, K, L, R, S, T, V and W; wherein X7005 is R or W;wherein X7006 is an amino acid selected from the group consisting of F,H, I, K, L, R, V and W; wherein X7007 is an amino acid selected from thegroup consisting of Orn, homoK, C, Hcy, Dap and K, preferably selectedfrom the group consisting of C and Hcy; wherein X7008 is an amino acidselected from the group consisting of A, G, R, S and T; wherein X7009 isan amino acid selected from the group consisting of a, A, I, K, L, M, m,Moo, Nle, p, R, Sem and V; wherein X7010 is an amino acid selected fromthe group consisting of A, G, I, K, L, P, R, S, T and V; wherein X7011is an amino acid selected from the group consisting of D, E, G, S and T;wherein X7012 is an amino acid selected from the group consisting of A,a, D, d, E, e, F, f, G, I, K, k, L, l, M, m, Moo, Nle, nle, P, p, R, r,S, s, Sem, T, t, V, v, W and w; wherein X7013 is an amino acid selectedfrom the group consisting of A, C, C(NEM), Con, Con(Meox), D, d, E, e,Eag, F, G, I, K, L, N, R, S, s, T, V and W; wherein X7014 is an aminoacid selected from the group consisting of A, D, E, F, G, I, K, L, M, R,S, T, V and W; wherein X7015 is an amino acid selected from the groupconsisting of A, D, E, F, G, I, K, L, M, Nle, R, S, T, V and W; whereinX7016 is an amino acid selected from the group consisting of A, D, E, F,I, K, L, M, Moo, Nle, R, S, Sem, T, V, W and Y; wherein X7017 is anamino acid selected from the group consisting of A, D, E, F, G, I, K, L,R, S, T, V, W and Y; wherein X7018 is an amino acid selected from thegroup consisting of C and D, preferably C; wherein X7019 is an aminoacid selected from the group consisting of A, F, I, L, S, T, V and W;wherein X7020 is an amino acid selected from the group consisting of Fand W; wherein X7021 is an amino acid selected from the group consistingof I, L and V; wherein X7022 is an amino acid selected from the groupconsisting of A, D, E, F, G, I, K, L, P, R, S, T, V and W; wherein X7023is either present or absent, whereby in case X7023 is present it is anamino acid selected from the group consisting of A, C, C(NEM), Con,Con(Meox), D, E, Eag, F, G, I, K, L, R, S, T, V, W and Y; and whereinthe peptide comprises as a cyclic structure generated by a linkagebetween X7007 and X7018, whereby degradation of TFPI by the serineprotease is inhibited.

41. The method of paragraph 40, wherein X7001 is an amino acid selectedfrom the group consisting of A, D, F, G, H, K, L and S; wherein X7002 isan amino acid selected from the group consisting of H, F, M and R;wherein X7003 is an amino acid selected from the group consisting of Fand Y; wherein X7004 is K; wherein X7005 is W; wherein X7006 is an aminoacid selected from the group consisting of F and H; wherein X7007 is C;wherein X7008 is an amino acid selected from the group consisting of A,G and S; wherein X7009 is an amino acid selected from the groupconsisting of M, Sem and V; wherein X7010 is an amino acid selected fromthe group consisting of K, P and R; wherein X7011 is D; wherein X7012 isan amino acid selected from the group consisting of F, L, l, M and Sem;wherein X7013 is an amino acid selected from the group consisting of D,G, K and S; wherein X7014 is G; wherein X7015 is an amino acid selectedfrom the group consisting of I and T; wherein X7016 is an amino acidselected from the group consisting of D, F, M, Sem and Y; wherein X7017is an amino acid selected from the group consisting of S and T; whereinX7018 is C; wherein X7019 is an amino acid selected from the groupconsisting of A and V; wherein X7020 is W; wherein X7021 is V; whereinX7022 is an amino acid selected from the group consisting of F, L, K, R,P and W; wherein X7023 is either present or absent, whereby in caseX7023 is present it is an amino acid sequence selected from the groupconsisting of A, D, F, M, S and Y; and wherein the peptide comprises asa cyclic structure generated by a linkage between X7007 and X7018.

42. The method of paragraph 40 or paragraph 41, wherein the peptide ispart of a peptide complex that further comprises a peptide comprisingthe structure of formula (XIII):

X6001-X6002-X6003-X6004-X6005-X6006-X6007-X6008-X6009-X6010-X6011-X6012-X6013-X6014-X6015-X6016-X6017-X6018-X6019-X6020(XIII) (SEQ ID NO: 3153);

wherein X6001 is an amino acid selected from the group consisting of F,L, M, Y, 1Ni, Thi, Bta, Dopa, Bhf, C, D, G, H, I, K, N, Nmf, Q, R, T, V,and W; wherein X6002 is an amino acid selected from the group consistingof Q, G, and K; wherein X6003 is an amino acid selected from the groupconsisting of C, D, E, M, Q, R, S, T, Ede(O), Cmc, A, Aib, Bhs, F, G, H,I, K, L, N, P, V, W and Y; wherein X6004 is an amino acid selected fromthe group consisting of Aib, E, G, I, K, L, M, P, R, W, Y, A, Bhk, C, D,F, H, k, N, Nmk, Q, S, T and V; wherein X6005 is an amino acid selectedfrom the group consisting of a, A, Aib, C, D, d, E, G, H, K, k, M, N,Nmg, p, Q, R, NpropylG, aze, pip, tic, oic, hyp, nma, Ncg, Abg, Apg,thz, dtc, Bal, F, L, S, T, V, W and Y; wherein X6006 is an amino acidselected from the group consisting of A, C, C(NEM), D, E, G, H, K, M, N,Q, R, S, V, Cit, C(Acm), Nle, I, Ede(O), Cmc, Ed, Eea, Eec, Eef, Nif,Eew, Aib, Btq, F, I, L, T, W and Y; wherein X6007 is an amino acidselected from the group consisting of I, V, T, Chg, Phg, Tle, A, F, G,I, K, L, Nmv, P, Q, S, W and Y; wherein X6008 is an amino acid selectedfrom the group consisting of F, H, 1Ni, 2Ni, Pmy, Y, and W; whereinX6009 is an amino acid selected from the group consisting of Aib, V,Chg, Phg, Abu, Cpg, Tle, L-2-amino-4,4,4-trifluorobutyric acid, A, f, I,K, S, T and V; wherein X6010 is an amino acid selected from the groupconsisting of A, C, D, d, E, F, H, K, M, N, P, Q, R, S, T, V, W, Y, Nmd,C(NEM), Aib, G, I, L and Nmf; wherein X6011 is an amino acid selectedfrom the group consisting of A, a, G, p, Sar, c, hcy, Aib, C, K, G andNmg; wherein X6012 is an amino acid selected from the group consistingof Y, Tym, Pty, Dopa and Pmy; wherein X6013 is an amino acid selectedfrom the group consisting of Aib, C, F, 1Ni, Thi, Bta, A, E, G, H, K, L,M, Q, R, W and Y; wherein X6014 is an amino acid selected from the groupconsisting of A, Aib, C, C(NEM), D, E, K, L, M, N, Q, R, T, V, Hcy, Bhe,F, G, H, I, P, S, W and Y; wherein X6015 is an amino acid selected fromthe group consisting of R, (omega-methyl)-R, D, E and K; wherein X6016is an amino acid selected from the group consisting of L, Hcy, Hle andAml; wherein X6017 is an amino acid selected from the group consistingof A, a, Aib, C, c, Cha, Dab, Eag, Eew, H, Har, Hci, Hle, I, K, L, M,Nle, Nva, Opa, Orn, R, S, Deg, Ebc, Eca, Egz, Aic, Apc, Egt,(omega-methyl)-R, Bhr, Cit, D, Dap, E, F, G, N, Q, T, V, W and Y;wherein X6018 is an amino acid selected from the group consisting of A,Aib, Hcy, hcy, C, c, L, Nle, M, N, R, Bal, D, E, F, G, H, I, K, Q, S, T,V, W and Y; wherein X6019 is an amino acid selected from the groupconsisting of K, R, Har, Bhk and V; and wherein X6020 is an amino acidselected from the group consisting of K, L, Hcy, Aml, Aib, Bhl, C, F, G,H, I, Nml, Q, R, S, T, V, W and Y.

43. The method of any one of paragraphs 40-43, wherein the protease iselastase, thrombin, plasmin, FXa, or chymase.

DESCRIPTION OF THE FIGURES

FIG. 1 is an illustration of the blood coagulation cascade.

FIG. 2 is an illustration of the secondary structure of Tissue FactorPathway Inhibitor-1.

FIG. 3 is an illustration of the formation of a quaternary complexcomprising Tissue Factor, Factor Xa (FXa), Factor VIIa (FVIIa), andTFPI.

FIG. 4 is a listing of amino acid sequences of various TFPI-bindingpeptides (e.g., TFPI-inhibitory peptides) denoting amino acidsubstitutions (bolded and underlined) in reference to peptide JBT0293.

FIG. 5 is an illustration of mRNA display selection of TFPI-bindingpeptides (e.g., TFPI-inhibitory peptides).

FIG. 6A is an illustration of the EC₅₀ binding ELISA and FIG. 6B is anillustration of the IC₅₀ ELISA described in Example 1.

FIG. 7 is a binding ELISA curve comparing % OD (y-axis) andconcentration [nM] (x-axis) for biotinylated peptide JBT0132.

FIGS. 8A-8D are competition ELISA curves comparing % OD (y-axis) andconcentration [nM] (x-axis) for exemplary peptides of the invention.

FIGS. 9A and 9B are sensorgrams plotting RU (y-axis) against time inseconds (x-axis) for peptides JBT0120 and JBT0132.

FIGS. 10A and 10B are sensorgrams plotting RU (y-axis) against time inseconds (x-axis) for peptide JBT0120 interaction with Tissue FactorPathway Inhibitor-1 and Tissue Factor Pathway Inhibitor-2.

FIGS. 11A and 11B are graphs comparing amount of thrombin generated (nM)(y-axis) and time in minutes (x-axis) for peptide JBT0120 and peptideJBT0132 in a plasma-based assay.

FIGS. 12A-12O, 13A-13D, 14A-14E, 15A-15D, 16A-16F, 17A-17B, and 18A-18Eare tables listing the amino acid sequences of various TFPI-bindingpeptides; EC₅₀ and percent inhibition of TFPI observed in the FXainhibition assay; EC₅₀ and percent inhibition of TFPI observed in theextrinsic tenase inhibition assay; and FEIBA, Factor VIII (FVIII)Immunate, or Factor IX (FIX) equivalent activities (mU/mL) inplasma-based assays. “*” denotes negative controls.

FIGS. 19A-19B, 20, and 21 are tables listing the results from BIAcoreanalysis of several TFPI-binding peptides. “*” denotes negativecontrols.

FIGS. 22A-22L, 23A-23K, 24A-24C, 25A-25L, 26A-26F, 27A-27C, 28A-28L,29A-29F, and 30A-30D are tables listing the amino acid sequences ofvarious TFPI-binding peptides; EC₅₀ and percent inhibition of TFPIobserved in the FXa inhibition assay; EC₅₀ and percent inhibition ofTFPI observed in the extrinsic tenase inhibition assay; and FEIBA, FVIIIImmunate, or FIX equivalent activities (mU/mL) in plasma-based assays.“*” denotes negative controls.

FIG. 31 is a graph comparing a pharmacokinetic characteristic(concentration of peptide (y-axis) versus time after administration(x-axis)) of a PEGylated TFPI-binding peptide to the pharmacokineticcharacteristic of same peptide lacking PEG. The peptides wereadministered intravenously to C57B16 mice at a dose of 10 mg/kg. Threebiological samples were analyzed for the presence of peptide at eachtime point.

FIGS. 32A-32AM, 33, 34A-34J, 35, 36A-36Q, 37, 38A-38B, and 39 are tableslisting the amino acid sequences and IC₅₀ or EC₅₀ values of variouspeptides of the invention. “*” denotes negative controls.

FIG. 40 is a graph illustrating a pharmacokinetic characteristic(concentration of peptide (nM) (y-axis) versus time after administration(minutes) (x-axis)) of a PEGylated TFPI-binding peptide followingsubcutaneous administration to mice at a dose of 10 mg/kg.

FIG. 41 is a graph correlating the amount of thrombin generated (nM)(y-axis) with time (minutes) (x-axis) for peptide JBT1855 in aplasma-based assay of hemophilia A patient plasma.

FIG. 42 is a graph illustrating the amount of blood loss (μl; y-axis)observed following a nail-clip in mice treated with JBT-1855(intravenous or subcutaneous administration), anti-TFPI antibody(intravenous administration), or vehicle (intravenous administration)(x-axis).

FIG. 43 is a graph plotting TFPI160 amino acid residue (x-axis) againstthe chemical shift differences of HSQC signals for free TFPI160 andTFPI160 bound to JBT0303 (y-axis).

FIG. 44 is a ribbon model of the secondary structure of TFPIillustrating regions of chemical shift changes of HSQC signals whenTFPI160 is complexed to JBT0303 compared to uncomplexed (free) TFPI160.

FIG. 45 is a graph plotting TFPI160 amino acid residue (x-axis) againstthe chemical shift differences of HSQC signals for free TFPI160 andTFPI160 bound to JBT0122 (y-axis).

FIG. 46 is a ribbon model of the secondary structure of TFPI proteinillustrating regions of chemical shift changes of HSQC signals whenTFPI160 is complexed to JBT0122 compared to uncomplexed (free) TFPI160.

FIG. 47 is a table listing assignments for the carbonyl carbon (C), thealpha carbon (CA), the beta carbon (CB), the amide proton (H), and theamide nitrogen (N) of JBT0788 based on HSQC, HNCACB, HNCA, HNCO and HNNspectra.

FIG. 48 is a ribbon model of the secondary structure of free JBT0788.

FIG. 49 is a table listing assignments for the carbonyl carbon (C), thealpha carbon (CA), the beta carbon (CB), the amide proton (H), and theamide nitrogen (N) of JBT0788 complexed with TFPI160 based on HSQC,HNCACB, HNCA, HCCOCA, and HNCO spectra.

FIG. 50 is a ribbon model of the secondary structure of JBT0788 whencomplexed with TFPI160.

FIG. 51 is a table listing assignments for the carbonyl carbon (C), thealpha carbon (CA), the beta carbon (CB), the amide proton (H), and theamide nitrogen (N) of JBT0616 based on HSQC, HNCACB, and HNN spectra.

FIG. 52 is a ribbon model of the secondary structure of free JBT0616.

FIG. 53 is a table listing assignments for the carbonyl carbon (C), thealpha carbon (CA), the beta carbon (CB), the amide proton (H), and theamide nitrogen (N) of JBT0616 complexed with TFPI based on HSQC, HNCO,HNCA, and HNCOCA spectra.

FIG. 54 is a ribbon model of the secondary structure of JBT0616 whencomplexed with TFPI160.

FIG. 55 is a ribbon structure of the energetically minimized best modelof KD1 (residues 22-79) in complex with JBT0303 with residues proposedto drive the protein-protein interaction displayed as sticks. Italicizedand underlined residues belong to JBT0303; the remaining residues belongto KD1 of TFPI.

FIG. 56 is a rotational thromboelastogram correlating sample elasticity(mm) with time in seconds (s) for JBT2317.

FIG. 57 is a rotational thromboelastogram correlating sample elasticity(mm) with time in seconds (s) for JBT2329.

FIG. 58 is an illustration of a computing device.

FIG. 59 is an illustration of a three dimensional (3D) model of a KD1protein.

FIG. 60 is an illustration of a 3D model of a TFPI-binding peptide.

FIG. 61 is an illustration of a method of modeling protein and peptideinteraction.

FIG. 62A-62F is a table listing the amino acid sequences and IC₅₀ orEC₅₀ values of various peptides of the invention. Designation “n.a.” is“not analyzed.” Progression curve data were obtained using the FXainhibition assay described in Example 3 with recombinant human fulllength TFPI. Assay concentration of progression curve assay was 0.0025%(0.1% Tween80 used in peptide dilution buffer).

FIG. 63 is a graph correlating concentration of peptides JBT2325-JBT2329(nM) (y-axis) with time following intravenous administration (hours)(x-axis). Peptides comprising higher weight PEG moieties exhibited aprolonged in vivo half life in mice. Each time point is represented bythe mean of three independent samples quantified by ELISA.

FIG. 64A-64C are graphs correlating concentration of peptides JBT2401,JBT2404 and JBT2410 (nM) (y-axis) with time following intravenousadministration (hours) (x-axis). Each time point is represented by themean of three independent samples quantified by ELISA. Solid circlessymbolize intravenous data, solid triangles symbolize subcutaneous data.

FIG. 65A-65B is a table listing the amino acid sequences of variouspeptides of the invention.

FIGS. 66A and 66B depicts IC₅₀ curves of TFPI-binding peptides JBT1837(SEQ ID NO: 1044) and JBT1857 (SEQ ID NO: 178) and peptide complexJBT2547 (SEQ ID NO: 4260) using two tracers: JBT2271 (SEQ ID NO: 4033),a biotinylated derivative of JBT1857 (FIG. 66A), and JBT2316 (SEQ ID NO:1313), a biotinylated derivative of JBT1837 (FIG. 66B).

FIG. 67 is a bar graph illustrating the results of a K_(off) assay withJBT2547 (SEQ ID NO: 4260) and JBT1857 (SEQ ID NO: 178) (X-axis)presented as % inhibition of TFPI binding by the tracer peptide, JBT2271(SEQ ID NO: 4033) (Y-axis).

FIGS. 68A-F illustrate the results of FXa inhibition assays using aTFPI-binding peptide complex. FIGS. 68A-68D are graphs correlatingconcentration of JBT1837 (triangles; SEQ ID NO: 1044), JBT1857 (circles;SEQ ID NO: 178), JBT2547 (diamonds; SEQ ID NO: 4260), andJBT1837+JBT1857 (squares) (μM) (X-axis) with percent inhibition of TFPIin a FXa inhibition assay performed with 0.5 nM full length human TFPI(FIG. 68A), 0.5 nM human TFPI 1-160 (FIG. 68B), 0.5 nM murine TFPI 1-160(FIG. 68C), or 0.5 nM cynomologus money TFPI 1-160 (FIG. 68D). FIG. 68Eillustrates TFPI inhibition by JBT2547 at increasing TFPI concentrations(0.1 to 10 nM from left to right) in a FXa inhibition assay. Data pointswere fitted by a sigmoidal dose response equation resulting in EC₅₀s(nM) and maximal inhibition (%). FIG. 68F is a graph correlating TFPIinhibition (%) by JBT2547 (diamonds), JBT2548 (circles), JBT1837(triangles) and a combination of JBT1837 and JBT1857 (squares) (1 μMpeptide) in the presence of increasing full length human TFPIconcentrations.

FIG. 69 illustrates the results of extrinsic tenase inhibition assaysusing a TFPI-binding peptide complex. FIG. 69A is a graph correlatingconcentration of JBT1837 (triangles; SEQ ID NO: 1044), JBT1857 (circles;SEQ ID NO: 178), JBT2547 (diamonds; SEQ ID NO: 4260), andJBT1837+JBT1857 (squares) (μM) (X-axis) with percent inhibition of TFPIin an extrinsic tenase inhibition assay performed with 0.063 nM fulllength human TFPI. FIG. 69B illustrates TFPI inhibition by JBT2547 atincreasing TFPI concentrations (0.031 to 10 nM from left to right) in anextrinsic tenase inhibition assay. Data points were fitted by asigmoidal dose response equation resulting in EC₅₀s (nM) and maximalinhibition (%). FIG. 69C is a graph correlating percent maximuminhibition of TFPI (Y-axis) mediated by JBT1837 (triangles; SEQ ID NO:1044), JBT2548 (circles; SEQ ID NO: 4261), JBT2547 (diamonds; SEQ ID NO:4260), and JBT1837+JBT1857 (squares) (1 μM) with concentration of fulllength human TFPI used in the extrinsic tenase inhibition assay. FIG.69D is a graph correlating EC₅₀ of JBT1837 (triangles; SEQ ID NO: 1044),JBT2548 (circles; SEQ ID NO: 4261), JBT2547 (diamonds); SEQ ID NO: 4260,and JBT1837+JBT1857 (squares) (1 μM) with concentration (nM) of fulllength human TFPI used in the extrinsic tenase inhibition assay. EC₅₀swere calculated by fitting of peptide concentrations response atincreasing TFPI concentrations vs. full length TFPI concentrations.

FIGS. 70A-70C illustrate the results of a thrombin generation assayperformed in FVIII-inhibited plasma. The figures correlate peak FactorIIa (thrombin) generation (nM) (Y-axis) with the concentration (nM) ofJBT1837 (triangle), JBT1857 (circle), a combination of JBT1837+JBT1857(square), and JBT2547 (diamond) (X-axis) in the presence of 1.25 nMflTFPI (FIG. 70A), 3.75 nM flTFPI (FIG. 70B), and 10 nM flTFPI (FIG.70C). Dotted line—pooled normal plasma (PNP); solid line-FVIII-inhibitedplasma (PNP+aFVIII).

FIG. 71A-71B are graphs correlating percent inhibition of TFPI (Y-axis)achieved at various concentrations (X-axis) of JBT2547, JBT1837, and1857 (FIG. 71A) or JBT2547 (diamonds), JBT1837 (triangles),JBT1837+JBT1857 (squares), and JBT2548 (circles) (FIG. 71B) in acell-based extrinsic tenase assay.

FIGS. 72A-72C illustrate the procoagulant effect of increasingconcentrations (10, 100, 1000 nM) of JBT2547 (FIG. 72A), JBT1837 (FIG.72B), or JBT1857 (FIG. 72C) in FVIII-inhibited whole blood in absence ofadditional external human full length TFPI (open circles) and presenceof increasing amounts of external full length TFPI (2 nM, closedtriangles; 10 nM, closed squares). Clot times of FVIII-inhibited wholeblood and normal whole blood are given as reference. +=no clot timeachieved.

FIG. 73 is a chart listing non-limiting examples of nucleophilic orelectrophilic reactive groups for peptide linkage.

FIG. 74 is a sequence alignment of TFPI KD1-KD2 from different species.Residues with a contact surface of 10-25 Å²=41, 56, 59, 60, 67, 71, 74,96, 106, 130, 132, 133, 136, 137, 142; residues with a contact surfaceof 26-60 Å²=75, 80, 82, 84, 85, 87, 145; residues with a contact surfaceof 61-100 Å²=63, 65, 131, 146; residues with a contact surface ofgreater than 100 Å²=83, 134.

FIG. 75 is a ribbon structure of the a model of KD1-KD2 in complex withJBT1857 and JBT1837.

FIG. 76A-76D is a table listing the amino acid sequences of variouspeptides of the invention.

FIG. 77 is a table listing the amino acid sequences of various peptidesof the invention.

FIGS. 78A-78AA is a table listing the amino acid sequences of variouspeptides of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides peptides and peptide complexes. In variousembodiments, the peptide or peptide complex binds Tissue Factor PathwayInhibitor-1 and, in some instances, blocks the inhibitory activity ofTissue Factor Pathway Inhibitor-1 (herein referred to as TFPI) withinthe blood coagulation cascade. Upon vascular injury, Tissue Factor (TF)complexes with Factor VIIa to form the “extrinsic complex” or “extrinsictenase complex,” which activates Factors IX and X (FIG. 1). TFPI is themain natural regulator of TF/FVIIa extrinsic complex activity and byextension, plays a role in controlling thrombin generation (Panteleev etal., Eur. J. Biochem., 249, 2016-2031 (2002)). TFPI is a 43 kDa serineprotease inhibitor comprising three Kunitz-type inhibitory domains (FIG.2). Kunitz domain 1 of TFPI binds FVIIa and Kunitz domain 2 binds FXa,enabling the inhibitor to form a quaternary FXa-TFPI-FVIIa-TF complexthat blocks activity of the TF/FVIIa extrinsic complex (FIG. 3). TFPIbinding of FXa also downregulates the common pathway of the coagulationcascade, during which FXa converts prothrombin to thrombin (Audu et al.,Anesth. Analg., 103(4), 841-845 (2006)). The invention provides, e.g.,TFPI-inhibitory peptides that block TFPI's inhibitory action on theblood coagulation cascade, thereby enhancing thrombin formation. In thecontext of the disclosure, any peptide encompassed by any of formulas(I) to (XIV) described herein and any TFPI-binding peptide describedherein is also referred to as “the peptide of the invention” and as “apeptide as described herein.”

The amino acid sequences of several TFPI-binding peptides are providedherein. Conventional amino acids are identified according to theirstandard, one-letter or three-letter codes, as set forth in Table 1.

TABLE 1 3-letter 1-letter codes code Amino acids Ala A Alanine Cys CCysteine Asp D Aspartic acid Glu E Glutamic acid Phe F Phenylalanine GlyG Glycine His H Histidine Ile I Isoleucine Lys K Lysine Leu L LeucineMet M Methionine Asn N Asparagine Pro P Proline Gln Q Glutamine Arg RArginine Ser S Serine Thr T Threonine Val V Valine Trp W Tryptophan TyrY Tyrosine

Examples of non-conventional amino acids and additional peptide buildingblocks are identified according to a three-letter code (with theexception of Ttds and Dopa, which are common four-letter abbreviations)found in Table 2. Additional building blocks designated by three-, four-or seven-number/letter designations or abbreviations also are listed inTable 2. The structures of some building blocks are depicted with anexemplary reagent for introducing the building block into a peptide(e.g., the structure provided for 2-naphthyl sulfonyl comprises achloride).

TABLE 2 Abbre- Name viation Structure Phenyl acetyl 374

2-Naphthyl sulfonyl 972

1-Naphthyl sulfonyl 973

3-Phenyl propionyl 1281

Hexanoyl 1525

3-Methyl butanoyl 3067

2-Methyl propionyl 4635

2-(Naphth-2-yl) acetyl 5963

N-(4-aminobutyl)-glycine Abg

2-aminobutyric acid Abu

2-Amino-isobutyric acid Aib

2-Aminoindane-2-carboxylic acid Aic

L-alpha-Methyl leucine Aml

Aminooxyacetic acid AOA

1-Amino-(4-N- piperidinyl)carboxylic acid Apc

N-(4-aminopropyl)-glycine Apg

D-Azetidine-2-carboxylic acid aze

β-Alanine Bal

β-Homoglutamatic acid Bhe

β-Homophenylalanine Bhf

β-Homolysine Bhk

β-Homoleucine Bhl

β-Homoasparagine Bhn

β-Homoglutamine Bhq

β-Homoarginine Bhr

β-Homoserine Bhs

β-Homotyrosine Bhy

β-Homoaspartic acid Bhd

β-Homovaline Bhv, Btl

β-Homoasparagin Bhn, Btq

L-3-Benzothienylalanine Bta

3-(Acetylamino-methylsulfanyl)-2- amino-propionic acid C(Acm)

Aminoethylthiol Cea

(S)-Cyclohexylalanine Cha

L-Cyclohexylglycine Chg

(S)-Citrullin Cit

Carboxymethylen cystein Cmc

N-ethylmaleiimido cysteine C(NEM)

8-amino-1,4-dioxa- spiro[4,5]decane-8-carboxylic acid Con

1-Amino-(4-methoximino)- cyclohexane-1-carboxlic acid Con(Meox)

L-Cyclopentylglycine Cpg

(S)-2,4-Diaminobutyric acid Dab

(S)-Diaminopropionic acid Dap

alpha,alpha-Diethylglycine Deg

5,5-Dimethyl-D-thiazolidine-4- carboxylic acid dtc

3,4-Dihydroxy-phenylalanine Dopa

(S)-2-Propargylglycine Eag

1-Amino-cyclopropane-1-carboxylic acid Ebc

1-Amino-cyclopentane-1-carboxylic acid Eca

Cys(3-propionic acid amide) Ecl

Sulfoxid of Carboxyethylcystein Ede(O)

Cys(5-methylen-2-oxazolidinon) Eea

Cys(1-methylen-1H-benzotriazol) Eec

Cys(3-methylen-2-benzothiazolinon) Eef

(S)-N(omega)-nitro-arginine Eew

alpha,alpha-Dibutylglycine Egt

1-amino-cyclohexane-1-carboxylic acid Egz

L-homophenylalanine Hfe

(S)-Homo-arginine Har

(S)-Homo-citrulline Hci

(S)-Homo-cysteine Hcy

D-Homo-cysteine hcy

(S)-2-Amino-5-methyl-hexanoic acid Hle

(S)-Homo-lysine Hly

ε-(Acetyl)-L-lysine K(Ac)

ε-(4-(p-Iodophenyl)butyryl))-L-lysine K(AlbuTag)

2-Amino-6-(2-aminooxy- acetylamino)-hexanoic acid K(AOA)

ε-(Myristyl)-L-lysine K(Myr)

ε-(Acetyl-Ttds)-L-lysine K(TtdsAc)

ε-(Myristyl-Ttds)-L-lysine K(TtdsMyr)

ε-(Myristyl-γ-glutamyl-Ttds)-L- lysine K(Ttds- γGlu-Myr)

ε-(Palmityl-Ttds)-L-lysine K(TtdsPal)

ε-(4-(Pentyl)- benzolsulfonamidyl)-L-lysine K(4PBSA)

4-Methoxyimino-pentanoic acid Meox-Lev

L-methionine-sulphone

1-Naphthylalanine 1Ni

2-Naphthylalanine 2Ni

N-(cyclohexyl)-glycine Ncg

4-Nitrophenyl alanine Nif

(S)-Norleucine Nle

(S)-N-Methylalanine Nma

(S)-N-Methyl-Aspartic acid Nmd

(S)-N-Methyl-glutamic acid Nme

(S)-N-Methyl-phenylalanine Nmf

N-Methyl-glycine Nmg

(S)-N-Methyl-lysine Nmk

(S)-N-Methyl-leucine Nml

N-Methyl-asparagine Nmn

(S)-N-Methyl-arginine Nmr

(S)-N-Methyl-serine Nms

(S)-N-Methyl-valine Nmv

(S)-N-Methyl-tyrosine Nmy

N-propyl glycine NpropylG

(S)-2-Amino-pentanoic acid Nva

(S)-2-Pyridyl-alanine Opa

Ornithine-(pyrazin-carboxylate) Opc

D-Octahydroindol-2-carboxylic acid oic

(S)-Ornithine Orn

Ethylidene-aminooxy-acetic acid Oxme- AOA

Palmitoyl Palm

L-Phenylglycin Phg

4-Phenyl-butyric acid PhPrCO

Polyethylene glycol PEG D-Pipecolic acid pip

L-Tyrosin(O-Methyl)—OH Pmy

L-Phosphotyrosine Pty

N-Methylglycine Sar

Selenomethionine Sem

L-2-Thienylalanine Thi

D-thiazolidine-4-carboxylic acid thz

1,2,3,4-L- tetrahydroisoquinolinecarboxylic acid Tic

L-alpha-t-Butylglycine Tle

(13-Amino-4,7,10-trioxa-tridecayl)- succinamic acid Ttds

Ttds- Maleimidopropionyl(EtSH))

3-Nitro-L-tyrosine Tym

Carboxyfluorescein FAM

[2-(2-Amino-ethoxy)-ethoxy]-acetic acid FA03202

3-{2-[2-(2-Amino-ethoxy)-ethoxy]- ethoxy}-propionic acid FA19203

3-(2-{2-[2-(2-Amino-ethoxy)-ethoxy]- ethoxy}-ethoxy)-propionic acidFA19204

3-[2-(2-{2-[2-(2-Amino-ethoxy)- ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionic acid FA19205

The amino acid sequences of the peptides provided herein are depicted intypical peptide sequence format, as would be understood by the ordinaryskilled artisan. For example, the three-letter code or one-letter codeof a conventional amino acid, or the three-, four-, orseven-number/letter code additional building blocks, indicates thepresence of the amino acid or building block in a specified positionwithin the peptide sequence. The code for each non-conventional aminoacid or building block is connected to the code for the next and/orprevious amino acid or building block in the sequence by a hyphen.Adjacent amino acids are connected by a chemical bond (typically anamide bond). The formation of the chemical bond removes a hydroxyl groupfrom the 1-carboxyl group of the amino acid when it is located to theleft of the adjacent amino acid (e.g., Hle-adjacent amino acid), andremoves a hydrogen from the amino group of the amino acid when it islocated on the right of the adjacent amino acid (e.g., adjacent aminoacid-Hle). It is understood that both modifications can apply to thesame amino acid and apply to adjacent conventional amino acids presentin amino acid sequences without hyphens explicitly illustrated. Where anamino acid contains more than one amino and/or carboxy group in theamino acid side chain, the 2- or 3-amino group and/or the 1-carboxygroup generally are used for the formation of peptide bonds. Fornon-conventional amino acids, a 3-letter code was used where the firstletter indicates the stereochemistry of the C-α-atom. For example, acapital first letter indicates that the L-form of the amino acid ispresent in the peptide sequence, while a lower case first letterindicating that the D-form of the correspondent amino acid is present inthe peptide sequence. When one-letter code is used, a lower case letterrepresents a D-amino acid, while an upper case letter represents anL-amino acid. Unless indicated to the contrary, the amino acid sequencesare presented herein in N- to C-terminus direction.

The C-termini of several TFPI-binding peptide sequences described hereinare explicitly illustrated by inclusion of an OH, NH₂, or anabbreviation for a specific terminating amine linked to the C-terminalamino acid code via a hyphen. The N-termini of several peptidesdescribed herein are explicitly illustrated by inclusion of a hydrogen(for a free N-terminus), or an abbreviation for a specific terminatingcarboxylic acid or other chemical group linked to the N-terminal aminoacid code via a hyphen.

The invention provides a peptide comprising the amino acid sequenceX₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁ (SEQ ID NO: 3109), wherein(using single letter codes for amino acids) X₇ is selected from thegroup consisting of L, P, K, S, W, V, N, and Q; X₈ is selected from thegroup consisting of L, R, N, F, and I; X₉ is selected from the groupconsisting of Y, V, P, and C; X₁₀ is selected from the group consistingof F, L, and G; X11 is selected from the group consisting of L, W, V, A,M, T, and S; X12 is selected from the group consisting of T, F, V, R, A,D, L, E, S, and Y; X13 is selected from the group consisting of I, M, G,Q, D, and R; X14 is selected from the group consisting of G, W, Y, L, M,and H; X15 is selected from the group consisting of N, P, F, H, K, andY; X16 is selected from the group consisting of M, D, E, V, G, and K;X17 is selected from the group consisting of G, I, R, S, T, and L; X18is selected from the group consisting of M, K, L, and I; X19 is selectedfrom the group consisting of Y, G, R, and S; X20 is selected from thegroup consisting of A, E, S, C, and Y; and X21 is selected from thegroup consisting of A, V, K, and E.

In addition to the core structure set forth above, X₇-X₂₁, otherstructures that are specifically contemplated are those in which one ormore additional amino acids are attached to the core structure (e.g.,linked to the N-terminus or the C-terminus of the amino acid sequenceX₇-X₂₁). Thus, the invention includes peptides comprising the corestructure and further comprising one or more N-terminal amino acid(s)comprising an amino acid sequence selected from the group consisting of:

X₆,

X₅X₆,

X₄X₅X₆,

X₃X₄X₅X₆ (SEQ ID NO: 3110),

X₂X₃X₄X₅X₆ (SEQ ID NO: 3111), and

X₁X₂X₃X₄X₅X₆ (SEQ ID NO: 3112);

wherein X₆ is directly linked to X₇ of the core structure amino acidsequence, and X₁ is selected from the group consisting of T and G; X₂ isselected from the group consisting of F and V; X₃ is selected from thegroup consisting of V, W, Y, and F; X₄ is selected from the groupconsisting of D, Q, and S; X₅ is selected from the group consisting ofE, T, N, and S; and X₆ is selected from the group consisting of R, H, K,and A. The peptide of the invention in one aspect comprises or consistsof the amino acid sequence QSKKNVFVFGYFERLRAK (SEQ ID NO: 1).

In another embodiment, the peptide of the invention comprising the corestructure comprises one or more C-terminal amino acid(s) comprising anamino acid sequence selected from the group consisting of:

X₂₂,

X₂₂X₂₃,

X₂₂X₂₃X₂₄,

X₂₂X₂₃X₂₄X₂₅ (SEQ ID NO: 3113),

X₂₂X₂₃X₂₄X₂₅X₂₆ (SEQ ID NO: 3114), and

X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇ (SEQ ID NO: 3115),

wherein X₂₂ is directly linked to X₂₁ of the core structure amino acidsequence, and X₂₂ is selected from the group consisting of Q, I, E, W,R, L, and N; X₂₃ is selected from the group consisting of L, V, M, andR; X₂₄ is selected from the group consisting of K, L, A, and Y; X₂₅ isF; X₂₆ is G; and X₂₇ is T.

In one aspect, the peptide of the invention comprises or consists of theamino acid sequence VIVFTFRHNKLIGYERRY (SEQ ID NO: 4). It is alsocontemplated that the peptide of the invention comprises additionalamino acids at both the N-terminus and the C-terminus of the corestructure. In this aspect, the peptide comprises or consists of theamino acid sequence TFVDERLLYFLTIGNMGMYAAQLKF (SEQ ID NO: 3),GVWQTHPRYFWTMWPDIKGEVIVLFGT (SEQ ID NO: 5), KWFCGMRDMKGTMSCVWVKF (SEQ IDNO: 6), or ASFPLAVQLHVSKRSKEMA (SEQ ID NO: 7).

The invention further includes peptides comprising the amino acidsequence X₃X₄X₅-F-X₇-NVF-X₁₁X₁₂-GY-X₁₅X₁₆-RLRAK-X₂₂ (SEQ ID NO: 2),wherein X₃ is Y or F; X₄ is Q or S; X₅ is N or S; X₇ is K, N, or Q; X₁₁is V, A, S, or T; X₁₂ is F, A, D, L, Q, S, or Y; X₁₅ is F, K, or Y; X₁₆is E or D; and X₂₂ is L or N.

In addition, the invention provides a peptide that binds TFPI, whereinthe peptide comprises the structure of formula (I):X1001-X1002-X1003-X1004-X1005-X1006-X1007-X1008-X1009-X1010-X1011-X1012-X1013-X1014-X1015-X1016-X1017-X1018-X1019-X1020(SEQ ID NO: 3116). In formula (I), X1001 is an amino acid selected fromthe group consisting of Bhf, C, D, F, G, H, I, K, L, M, N, Nmf, Q, R, T,V, W, and Y; X1002 is an amino acid selected from the group consistingof G, K, and Q; X1003 is an amino acid selected from the groupconsisting of A, Aib, Bhs, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, and Y; X1004 is an amino acid selected from the groupconsisting of, A, Aib, Bhk, C, D, E, F, G, H, I, K, k, L, M, N, Nmk, P,Q, R, S, T, V, W, and Y; X1005 is an amino acid selected from the groupconsisting of a, A, Aib, Bal, C, D, d, E, F, G, H, K, k, L, M, N, Nmg,p, Q, R, S, T, V, W, and Y; X1006 is an amino acid selected from thegroup consisting of A, Aib, Btq, C, D, E, F, G, H, I, K, L, M, N, Q, R,S T, V, W, and Y; X1007 is an amino acid selected from the groupconsisting of A, F, G, I, K, L, Nmv, P, Q, S, V, W, and Y; X1008 is anamino acid selected from the group consisting of F, H, K, W, and Y;X1009 is an amino acid selected from the group consisting of A, Aib, f,I, K, S, T, and V; X1010 is an amino acid selected from the groupconsisting of A, Aib, C, D, E, F, G, H, I, K, L, M, N, Nmf, P, Q, R, S,T, V, W, and Y; X1011 is an amino acid selected from the groupconsisting of Aib, C, K, G, and Nmg; X1012 is Y; X1013 is an amino acidselected from the group consisting of A, Aib, C, E, F, G, H, K, L, M, Q,R, W, and Y; X1014 is an amino acid selected from the group consistingof A, Aib, Bhe, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,and Y; X1015 is an amino acid selected from the group consisting of(omega-methyl)-R, D, E, K, and R; X1016 is L; X1017 is an amino acidselected from the group consisting of (omega-methyl)-R, A, Aib, Bhr, C,Cha, Cit, D, Dab, Dap, E, Eag, Eew, F, G, H, Har, Hci, Hle, I, K, L, M,N, Nle, Nva, Opa, Orn, Q, R, S, T, V, W, and Y; X1018 is an amino acidselected from the group consisting of A, Bal, C, D, E, F, G, H, I, K, L,M, N, Q, R, S, T, V, W, and Y; and X1019 is an amino acid selected fromthe group consisting of Bhk, K, R, and V. X1020 is either present orabsent in formula (I) (i.e., in some instances, the peptide of theinvention comprises the structureX1001-X1002-X1003-X1004-X1005-X1006-X1007-X1008-X1010-X1011-X1012-X1013-X1014-X1015-X1016-X1017-X1018-X1019(SEQ ID NO: 3116)). When X1020 is present, it is an amino acid selectedfrom the group consisting of Aib, Bhl, C, F, G, H, I, K, L, Nml, Q, R,S, T, V, W, and Y.

For example, the peptide of the invention comprises the structure offormula (I) wherein X1001 is an amino acid selected from the groupconsisting of C, F, I, K, L, Nmf, V, M, W, and Y; X1002 is Q; X1003 isan amino acid selected from the group consisting of A, C, D, E, H, K, M,I, N, Q, R, S, T, and V; X1004 is an amino acid selected from the groupconsisting of A, Aib, C, D, E, G, H, F, I, K, k, L, M, N, Nmk, P, Q, R,S, V, W, and Y; X1005 is an amino acid selected from the groupconsisting of a, A, Aib, Bal, C, d, E, D, F, G, H, K, k, L, M, N, Nmg,p, Q, R, S, T, and Y; X1006 is an amino acid selected from the groupconsisting of A, Btq, C, D, G, I, K, H, L, M, N, Q, R, S, V, and Y;X1007 is an amino acid selected from the group consisting of I, K, L, Q,V, and Y; X1008 is an amino acid selected from the group consisting ofF, H, and Y; X1009 is an amino acid selected from the group consistingof f, I, and V; X1010 is an amino acid selected from the groupconsisting of A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, and Y;X1011 is an amino acid selected from the group consisting of G and Nmg;X1012 is Y; X1013 is an amino acid selected from the group consisting ofAib, C, F, H, L, W, and Y; X1014 is an amino acid selected from thegroup consisting of A, Aib, Bhe, C, D, E, H, I, K, L, M, N, Q, R, S, T,V, W, and Y; X1015 is an amino acid selected from the group consistingof E and R; X1016 is L; X1017 is an amino acid selected from the groupconsisting of (omega-methyl)-R, A, Aib, Bhr, C, Cha, Cit, Dab, Dap, Eag,Eew, F, H, Har, Hci, Hle, I, K, L, M, N, Nle, Nva, Opa, Orn, R, S, T, V,and Y; X1018 is an amino acid selected from the group consisting of A,C, D, E, F, I, K, L, M, N, Q, R, V, and W; X1019 is an amino acidselected from the group consisting of K and R; and X1020 is an aminoacid selected from the group consisting of Aib, Bhl, F, K, L, R, and W(when X1020 is present in the peptide).

In one aspect, the peptide of the invention comprises the structure offormula (I) wherein X1001 is an amino acid selected from the groupconsisting of F, L, Y, and M; X1002 is Q; X1003 is an amino acidselected from the group consisting of M, Q, R, S, T, and C; X1004 is anamino acid selected from the group consisting of Aib, K, L, P, R, E, G,I, Y, M, and W; X1005 is an amino acid selected from the groupconsisting of a, Aib, D, d, G, H, K, k, N, Nmg, p, Q, R, A, E, C, and M;X1006 is an amino acid selected from the group consisting of A, C, D, G,H, K, N, Q, R, S, and M; X1007 is an amino acid selected from the groupconsisting of I and V; X1008 is an amino acid selected from the groupconsisting of F, H, and Y; X1009 is V; X1010 is an amino acid selectedfrom the group consisting of A, D, E, K, M, N, Q, R, F, H, P, S, V, W,and Y; X1011 is G; X1012 is Y; X1013 is C or F; X1014 is an amino acidselected from the group consisting of A, C, D, E, K, L, M, N, Q, R, T,V, and Aib; X1015 is R; X1016 is L; X1017 is an amino acid selected fromthe group consisting of A, Aib, C, Cha, Dab, Dap, Eag, Eew, H, Har, Hci,Hle, K, Nle, Nva, Opa, Orn, R, I, L, S, and M; X1018 is an amino acidselected from the group consisting of A, L, N, M, and R; X1019 is K; andX1020 is K or L.

When amino acid X1020 is absent from formula (I), the peptide of theinvention in one aspect further comprises amino acid X1000 at theN-terminus of formula (I), such that the peptide comprises or consistsof the structure of formula (II):X1000-X1001-X1002-X1003-X1004-X1005-X1006-X1007-X1008-X1009-X1010-X1011-X1012-X1013-X1014-X1015-X1016-X1017-X1018-X1019(II) (SEQ ID NO: 3122). When X1000 is present in the peptide, X1000 isan amino acid selected from the group consisting of A, E, and P, whilethe amino acids of X1001-X1019 are as defined above.

In an additional aspect, the TFPI-binding peptide of the inventioncomprises the structure of formula (III):X1001-Q-X1003-X1004-X1005-X1006-I/V-X1008-V-X1010-G-Y-C/F-X1014-R-L-X1017-X1018-K-K/L(III) (SEQ ID NO: 3117). As used herein, amino acid designationsseparated by “I” refer to alternative amino acid residues at theindicated position. For example, with respect to formula (III), theamino acid residue at position 7 is isoleucine or valine. X1001, X1003,X1004, X1005, X1006, X1008, X1010, X1014, X1017 and X1018 in formula(III) are each independently selected from any amino acid. For example,in formula (III), X1001 is optionally an amino acid selected from thegroup consisting of Bhf, C, D, F, G, H, I, K, L, M, N, Nmf, Q, R, T, V,W, and Y, such as an amino acid selected from the group consisting of C,F, I, K, L, Nmf, V, M, W, and Y (e.g., an amino acid selected from thegroup consisting of F, L, Y and M); X1003 is optionally an amino acidselected from the group consisting of A, Aib, Bhs, C, D, E, F, G, H, I,K, L, M, N, P, Q, R, S, T, V, W, and Y, such as an amino acid selectedfrom the group consisting of A, C, D, E, H, K, M, I, N, Q, R, S, T, andV (e.g., the amino acid is M, Q, R, S, T or C); X1004 is optionally anamino acid selected from the group consisting of, A, Aib, Bhk, C, D, E,F, G, H, I, K, k, L, M, N, Nmk, P, Q, R, S, T, V, W, and Y, such as anamino acid selected from the group consisting of A, Aib, C, D, E, G, H,F, I, K, k, L, M, N, Nmk, P, Q, R, S, V, W, and Y (e.g., an amino acidselected from the group consisting of Aib, K, L, P, R, E, G, I, Y, M,and W); X1005 is optionally an amino acid selected from the groupconsisting of a, A, Aib, Bal, C, D, d, E, F, G, H, K, k, L, M, N, Nmg,p, Q, R, S, T, V, W, and Y, such as an amino acid selected from thegroup consisting of a, A, Aib, Bal, C, d, E, D, F, G, H, K, k, L, M, N,Nmg, p, Q, R, S, T, and Y (e.g., the amino acid is a, Aib, D, d, G, H,K, k, N, Nmg, p, Q, R, A, E, C, or M); X1006 is optionally an amino acidselected from the group consisting of A, Aib, Btq, C, D, E, F, G, H, I,K, L, M, N, Q, R, S, T, V, W, and Y, such as an amino acid selected fromthe group consisting of A, Btq, C, D, G, I, K, H, L, M, N, Q, R, S, V,and Y (e.g., an amino acid selected from the group consisting of A, C,D, G, H, K, N, Q, R, S, and M); X1008 is optionally an amino acidselected from the group consisting of F, H, K, W, and Y, such as anamino acid selected from the group consisting of F, H, and Y; X1010 isoptionally an amino acid selected from the group consisting of A, Aib,C, D, E, F, G, H, I, K, L, M, N, Nmf, P, Q, R, S, T, V, W, and Y, suchas an amino acid selected from the group consisting of A, D, E, F, G, H,K, L, M, N, P, Q, R, S, T, V, W, and Y (e.g., an amino acid selectedfrom the group consisting of A, D, E, K, M, N, Q, R, F, H, P, S, V, W,and Y); X1014 is optionally an amino acid selected from the groupconsisting of A, Aib, Bhe, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, and Y, such as an amino acid selected from the group consistingof A, Aib, Bhe, C, D, E, H, I, K, L, M, N, Q, R, S, T, V, W, and Y(e.g., A, C, D, E, K, L, M, N, Q, R, T, V, or Aib); X1017 is optionallyan amino acid selected from the group consisting of (omega-methyl)-R, A,Aib, Bhr, C, Cha, Cit, D, Dab, Dap, E, Eag, Eew, F, G, H, Har, Hci, Hle,I, K, L, M, N, Nle, Nva, Opa, Orn, Q, R, S, T, V, W, and Y, such as anamino acid selected from the group consisting of (omega-methyl)-R, A,Aib, Bhr, C, Cha, Cit, Dab, Dap, Eag, Eew, F, H, Har, Hci, Hle, I, K, L,M, N, Nle, Nva, Opa, Orn, R, S, T, V, and Y (e.g., an amino acidselected from the group consisting of A, Aib, C, Cha, Dab, Dap, Eag,Eew, H, Har, Hci, Hle, K, Nle, Nva, Opa, Orn, R, I, L, S, and M); and/orX1018 is optionally an amino acid selected from the group consisting ofA, Bal, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y, suchas an amino acid selected from the group consisting of A, C, D, E, F, I,K, L, M, N, Q, R, V, and W (e.g., an amino acid selected from the groupconsisting of A, L, N, M, and R).

In some embodiments, the peptide of the invention comprises one or moreadditional amino acid residues attached to the N- or C-terminus of theamino acid sequence. For example, the peptide comprising the structureof any one of formulas (I)-(III), in some embodiments, further comprisesone or more N-terminal amino acid(s) directly linked to X1001, whereinthe N-terminal amino acid(s) comprise the amino acid sequence selectedfrom the group consisting of X1000, X999-X1000, X998-X999-X1000,X997-X998-X999-X1000 (SEQ ID NO: 3123), X996-X997-X998-X999-X1000 (SEQID NO: 3124), X995-X996-X997-X998-X999-X1000 (SEQ ID NO: 3125),X994-X995-X996-X997-X998-X999-X1000 (SEQ ID NO: 3126),X993-X994-X995-X996-X997-X998-X999-X1000 (SEQ ID NO: 3127),X992-X993-X994-X995-X996-X997-X998-X999-X1000 (SEQ ID NO: 3128),X991-X992-X993-X994-X995-X996-X997-X998-X999-X1000 (SEQ ID NO: 3129),and X990-X991-X992-X993-X994-X995-X996-X997-X998-X999-X1000 (SEQ ID NO:3130). When the peptide comprises one or more N-terminal amino acids,X1000 is A or K; X999 is V or K; X998 is Q or K; X997 is L or K; X996 isR or K; X995 is G or K; X994 is V or K; X993 is G or K; X992 is S or K;X991 is K; and X990 is K.

In addition to the core structures set forth in formulas (I)-(III),other structures that are specifically contemplated are those in whichone or more additional amino acids are attached to the C-terminus of thecore structure directly linked to X1020. For example, the C-terminaladdition optionally comprises an amino acid sequence selected from thegroup consisting of X1021, X1021-X1022, X1021-X1022-X1023, andX1021-X1022-X1023-X1024 (SEQ ID NO: 3131), wherein X1021 is T or K;X1022 is S or K; and X1023 and X1024 are K.

The invention further includes a TFPI-binding peptide comprising orconsisting of an amino acid sequence having at least 60% identity (e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or 100% identity) to the amino acid sequenceAc-FQSK-Nmg-NVFVDGYFERL-Aib-AKL-NH2 (formula IV) (SEQ ID NO: 164). Insome instances, the peptide comprises or consists of the amino acidsequence of any one of formulas (I)-(III), as described herein. Theinvention also includes a peptide comprising or consisting of an aminoacid sequence selected from the group consisting of SEQ ID NOs: 8-978(e.g., a peptide comprising or consisting of the amino acid sequenceselected from the group consisting of SEQ ID NOs: 8-741 and 962-972(such as SEQ ID NOs: 8-741, 962-968, 971, or 972) and/or selected fromthe group consisting of 742-961 (such as SEQ ID NOs: 744-961) and/orselected from the group consisting of SEQ ID NOs: 973-978).

The invention includes peptides that comprise a cyclic structure. Inthis regard, the invention includes peptides comprising cyclicstructures within the peptide (e.g., one or more loops formed by linkagebetween amino acids other than the N- and C-terminal amino acids),peptides comprising a cyclic structure formed by the interaction of aterminal amino acid with an amino acid within the peptide sequence, andpeptides cyclized head to tail. The peptide may also be part of a largercyclic structure formed by surrounding additional amino acids orchemical substituents. The peptides of the invention, in some instances,comprise intramolecular disulfide bonds. In some embodiments, theintramolecular disulfide bonds are formed by cysteine residues. Peptidescomprising cyclic structures formed by non-cysteine residues, or anon-cysteine residue and a cysteine residue, also are provided. Forexample, in one embodiment, the inventive peptide comprises at least onenon-conventional amino acid or chemical moiety that mediatescyclization. Suitable non-conventional amino acids or chemical moietiesinclude, but are not limited to, FA19205, FA19204, FA19203, FA03202,Hcy, hcy, Cea, and c. The amino acids or moieties responsible forcyclization are sufficiently spaced apart to allow formation of a loopstructure, e.g., the amino acids or moieties are separated by two,three, four, five, six, seven, eight, or more residues.

In one aspect, the peptide comprising the structure of formulas(I)-(III) contains at least two cysteine residues (e.g., the peptidecontains two cysteine residues) that are spaced apart by at least threeamino acid residues such that the cysteines form an intramoleculardisulfide bond. In some instances, the cysteines are spaced apart bymore than three amino acid residues. For example, in the peptidecomprising the structure of formulas (I), (II), or (III), any two ofX1000, X1001, X1003, X1004, X1005, X1006, X1010, X1011, X1013, X1014,X1017, X1018, X1020 and X1021 are optionally cysteines capable offorming a disulfide bridge. Accordingly, in some aspects, the peptidecontains two cysteine residues: one of X1000, X1005, X1010 and X1014 iscysteine, and one of X1006, X1010, X1017 and X1021 is a cysteine. Theinvention contemplates all of the possible combinations of cysteinepairs, e.g., X1000 and X1006 are C; X1000 and X1010 are C; X1000 andX1017 are C; X1005 and X1017 are C; X1010 and X1017 are C; X1010 andX1021 are C; or X1014 and X1021 are C. Other exemplary cyclic peptidesof the invention include, e.g., JBT2441, JBT2450, JBT2466-JBT2469,JBT2489-JBT2495, JBT2497-JBT2499, and JBT2513-JBT2518 (SEQ ID NOs: 4159,4167, 4181-4184, 4204-4210, 4212-4214, and 4228-4233, respectively.).

The invention further provides a peptide that binds TFPI, the peptidecomprising the structure of formula (V):X2001-X2002-X2003-X2004-X2005-X20064-[X2007-X2008-X2009-X2010-X2011-X2012-X2013-X2014-X2015-X2016-X2017-X2018]-X2019-X2020-X2021-X2022-X2023(V) (SEQ ID NO: 3118), wherein the peptide forms a cyclic structuregenerated by a linkage, e.g., a disulfide bond, between X2007 and X2018(denoted as brackets within formula (V)). In formula (V), X2001, X2002,and X2023 are independently either present or absent. When present,X2001 is an amino acid selected from the group consisting of A, D, E, F,G, H, I, K, L, P, R, S, T, V, and W; X2002 an amino acid selected fromthe group consisting of A, D, E, F, G, H, I, K, L, M, P, R, S, T, V, andW; and X2023 is an amino acid selected from the group consisting of A,D, E, F, G, I, K, L, R, S, T, V, W, and Y. In addition, X2003 is anamino acid selected from the group consisting of A, F, I, K, L, R, S, T,V, W, and Y; X2004 is an amino acid selected from the group consistingof A, D, E, F, G, I, K, L, R, S, T, V, and W; X2005 is W; X2006 is anamino acid selected from the group consisting of F, H, I, K, L, R, V,and W; X2007 is an amino acid selected from the group consisting of C,Hcy, Dap, and K (e.g., C or Hcy); X2008 is an amino acid selected fromthe group consisting of A, G, R, S, and T; X2009 is an amino acidselected from the group consisting of a, A, I, K, L, M, m, Nle, p, R,Sem, and V; X2010 is an amino acid selected from the group consisting ofA, G, I, K, L, P, R, S, T, and V; X2011 is an amino acid selected fromthe group consisting of D, E, G, S, and T; X2012 is an amino acidselected from the group consisting of A, a, D, d, E, e, F, f, G, I, K,k, L, l, M, m, Nle, nle, P, p, R, r, S, s, Sem, T, t, V, v, W, and w;X2013 is an amino acid selected from the group consisting of A, D, d, E,e, F, G, I, K, L, R, S, s, T, V, and W; X2014 is an amino acid selectedfrom the group consisting of A, D, E, F, G, I, K, L, M, R, S, T, V, andW; X2015 is an amino acid selected from the group consisting of A, D, E,F, G, I, K, L, M, Nle, R, S, T, V, and W; X2016 is an amino acidselected from the group consisting of A, D, E, F, I, K, L, M, Nle, R, S,Sem, T, V, W, and Y; X2017 is an amino acid selected from the groupconsisting of A, D, E, F, G, I, K, L, R, S, T, V, W, and Y; X2018 is anamino acid selected from the group consisting of C and D (e.g., X2018 isC); X2019 is an amino acid selected from the group consisting of A, F,I, L, S, T, V, and W; X2020 is an amino acid selected from the groupconsisting of F and W; X2021 is an amino acid selected from the groupconsisting of I, L, and V; and X2022 is an amino acid selected from thegroup consisting of A, D, E, F, G, I, K, L, P, R, S, T, V, and W.

In some instances, in the peptide of the invention comprising thestructure of formula (V), X2001 is optionally an amino acid selectedfrom the group consisting of A, D, F, G, H, K, L, P, and S, such as anamino acid selected from the group consisting of A, D, F, G, H, K, L,and S (when X2001 is present); X2002 is optionally an amino acidselected from the group consisting of A, D, F, G, H, K, L, P, R, and S,such as an amino acid selected from the group consisting of A, F, H, K,L, M, R, and S (e.g., H, F, M or R) (when X2002 is present); X2003 isoptionally an amino acid selected from the group consisting of A, F, K,L, S, and Y, such as an amino acid selected from the group consisting ofF, S, and Y (e.g., F or Y); X2004 is optionally an amino acid selectedfrom the group consisting of A, D, F, G, K, L, and S (e.g., K); X2005 isoptionally W; X2006 is optionally an amino acid selected from the groupconsisting of F, H, K, and L (e.g., F or H); X2007 is optionally anamino acid selected from the group consisting of C and HcY (e.g., X2007is C); X2008 is optionally an amino acid selected from the groupconsisting of A, G, and S; X2009 is optionally an amino acid selectedfrom the group consisting of a, A, K, L, V, M, m, Nle, Sem, and p, suchas an amino acid selected from the group consisting of M, Nle, p, and V(e.g., M, Sem, or V); X2010 is optionally an amino acid selected fromthe group consisting of A, G, K, L, P, R, and S, such as an amino acidselected from the group consisting of A, K, L, P, R and S (e.g., K, P,or R); X2011 is optionally an amino acid selected from the groupconsisting of D, G, and S (e.g., D or S); X2012 is optionally an aminoacid selected from the group consisting of A, a, D, d, F, f, G, K, k, L,l, M, m, Nle, P, S, and s, such as an amino acid selected from the groupconsisting of D, d, F, f, G, K, k, L, l, M, Nle, P, S, and Sem (e.g., anamino acid selected from the group consisting of F, L, l, Sem, and M);X2013 is optionally an amino acid selected from the group consisting ofA, D, d, F, G, K, L, S, and s, such as an amino acid selected from thegroup consisting of A, D, F, G, K, L and S (e.g., D, G, K, or S); X2014is optionally an amino acid selected from the group consisting of D, F,G, K, L, and S (e.g., D or G); X2015 is optionally an amino acidselected from the group consisting of A, D, F, G, I, K, L, M, Nle, S,and T (e.g., I or T); X2016 is optionally an amino acid selected fromthe group consisting of D, F, K, L, M, Nle, S, and Y, such as an aminoacid selected from the group consisting of D, F, K, L, M, Nle, S, Sem,and Y (e.g., D, F, M, Sem, or Y); X2017 is optionally an amino acidselected from the group consisting of A, D, F, G, K, L, S, T, and Y(e.g., S or T); X2018 is optionally C; X2019 is optionally an amino acidselected from the group consisting of A, F, L, S, and V (e.g., A or V);X2020 is optionally an amino acid selected from the group consisting ofF and W (e.g., W); X2021 is optionally an amino acid selected from thegroup consisting of L and V (e.g., V); X2022 is optionally an amino acidselected from the group consisting of A, D, F, G, K, L, P, R, S, and W,such as an amino acid selected from the group consisting of A, F, G, K,L, P, R, S, and W (e.g., an amino acid selected from the groupconsisting of F, L, K, R, P, and W); and X2023 is optionally an aminoacid selected from the group consisting of A, D, F, G, K, L, M, S, andY, such as an amino acid selected from the group consisting of A, D, F,G, L M, S, and Y (e.g., an amino acid sequence selected from the groupconsisting of A, D, F, M, S and Y) (when X2023 is present).

The invention further includes a peptide that binds TFPI, wherein thepeptide comprises the structure of formula (VI):X2001-X2002-F/Y-K-W-F/H-[C-X2008-M/V-X2010-D-X2012-X2013-G-I/T-X2016-SIT-C]-A/V-W-V-X2022-X2023(VI) (SEQ ID NO: 3119). In the peptide comprising the structure offormula (VI), X2001, X2002 and X2023 are each independently present orabsent. If X2001, X2002, and/or X2023 are present, any of X2001, X2002and X2023 is independently selected from any amino acid. In addition,X2008, X2010, X2012, X2013, X2016, and X2022 are each independentlyselected from any amino acid.

In some aspects, in the peptide of formula (VI), X2001 is optionally anamino acid selected from the group consisting of A, D, E, F, G, H, I, K,L, P, R, S, T, V, and W, such as an amino acid selected from the groupconsisting of A, D, F, G, H, K, L, P, and S (e.g., an amino acidselected from the group consisting of A, D, F, G, H, K, L, and S) (whenX2001 is present); X2002 is optionally an amino acid selected from thegroup consisting of A, D, E, F, G, H, I, K, L, M, P, R, S, T, V, and W,such as an amino acid selected from the group consisting of A, D, F, G,H, K, L, M, P, R, and S (e.g., an amino acid selected from the groupconsisting of A, F, H, K, L, M, R, and S, such as H, F, M, or R) (whenX2002 is present); X2008 is optionally an amino acid selected from thegroup consisting of A, G, R, S, and T, such as an amino acid selectedfrom the group consisting of A, G, and S; X2010 is optionally an aminoacid selected from the group consisting of A, G, I, K, L, P, R, S, T,and V, such as an amino acid selected from the group consisting of A, G,K, L, P, R, and S (e.g., an amino acid selected from the groupconsisting of A, K, L, P, R, and S, such as K, P or R); X2012 isoptionally an amino acid selected from the group consisting of A, a, D,d, E, e, F, f, G, I, I, K, k, L, l, M, m, Nle, nle, P, p, R, r, S, s,Sem, T, t, V, v, W, and w, such as an amino acid selected from the groupconsisting of A, a, D, d, F, f, G, K, k, L, l, M, m, Nle, P, S, s, andSem (e.g., an amino acid selected from the group consisting of D, d, F,f, G, K, k, L, l, M, Nle, P, S, and Sem, such as F, L, l, Sem, or M);X2013 is optionally an amino acid selected from the group consisting ofA, D, d, E, e, F, G, I, K, L, R, S, s, T, V, and W, such as an aminoacid selected from the group consisting of A, D, d, F, G, K, L, S, and s(e.g., an amino acid selected from the group consisting of A, D, F, G,K, L, and S, such as D, G, K, or S); X2016 is optionally an amino acidselected from the group consisting of A, D, E, F, I, K, L, M, Nle, R, S,Sem, T, V, W, and Y, such as an amino acid selected from the groupconsisting of D, F, K, L, M, Nle, S, Sem, and Y (e.g., an amino acidselected from the group consisting of D, F, K, L, M, Nle, S, and Sem,such as F, Sem, or M); X2022 is optionally an amino acid selected fromthe group consisting of A, D, E, F, G, I, K, L, P, R, S, T, V, and W,such as an amino acid selected from the group consisting of A, D, F, G,K, L, P, R, S, and W (e.g., an amino acid selected from the groupconsisting of A, F, G, K, L, P, R, S, and W, such as F, L, K, R, P, orW); and/or X2023 is optionally an amino acid selected from the groupconsisting of A, D, E, F, G, I, K, L, R, M, S, T, V, W, and Y, such asan amino acid selected from the group consisting of A, D, F, G, K, L, M,S, and Y (e.g., an amino acid selected from the group consisting of A,D, F, G, L M, S, and Y, such as A, D, F, M, S, or Y) (when X2023 ispresent).

The TFPI-binding peptide of the invention, in one aspect, comprises anamino acid sequence having at least 60% identity (e.g., at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or 100% identity) to the sequence of formulaVII: Ac-FYYKWH[CGMRDMKGTMSC]AWVKF-NH2 (VII) (SEQ ID NO: 1040).Optionally, the peptide comprises or consists of the amino acid sequenceof formula (V)-(VII) as defined herein. The invention also includes apeptide comprising or consisting of the amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1001-1293 (e.g., a peptidecomprising or consisting of the amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1001-1212 and 1290-1291 (such as SEQ IDNOs: 1001-120, 1290, or 1291) and/or selected from the group consistingof SEQ ID NOs: 1213-1289 and/or selected from the group consisting of1292 and 1293). The invention also includes a peptide comprising (orconsisting of) the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1337-1355 and 4240-4268 (JBT0496, JBT1165,JBT2330-JBT2338, JBT2341, JBT2342, JBT2346-2348, JBT2356, JBT2457,JBT2538, JBT2519-JBT2523, JBT2527-JBT2529, JBT2531-JBT2534, JBT2537,JBT2539-JBT2541, JBT2543-JBT2555).

The invention further provides a TFPI-binding peptide comprising atleast amino acids 3-21 (X3003-X3021) of the structure of formula (VIII):X3001-X3002-X3003-X3004-X3005-X3006-X3007-X3008-X3009-X3010-X3011-X3012-X3013-X3014-X3015-X3016-X3017-X3018-X3019-X3020-X3021(VIII) (SEQ ID NO: 3120). In formula (VIII), X3001 and X3002 areindependently either present or absent in the peptide. If present, X3001is an amino acid selected from the group consisting of A, C, D, F, G, I,K, L, M, N, P, Q, R, S, T, W, E, H, and Y; and X3002 is an amino acidselected from the group consisting of A, C, D, F, H, K, M, N, P, R, S,T, W, Y, G, I, and L. In addition, X3003 is an amino acid selected fromthe group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, W, and Y; X3004 is an amino acid selected from the group consistingof A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, and P; X3005is an amino acid selected from the group consisting of C, D, F, G, H, I,K, L, M, N, P, R, S, T, V, W, and Y; X3006 is an amino acid selectedfrom the group consisting of A, W, C, K, P, R, and H; X3007 is an aminoacid selected from the group consisting of Q, A, C, F, G, H, I, K, L, N,R, S, T, W, and Y; X3008 is an amino acid selected from the groupconsisting of A, C, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, Y, and I;X3009 is an amino acid selected from the group consisting of A, C, F, G,H, I, L, M, R, S, T, V, W, Y, and K; X3010 is an amino acid selectedfrom the group consisting of A, C, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, and Y; X3011 is an amino acid selected from the groupconsisting of A, G, I, K, L, M, N, Q, R, S, T, V, W, Y, C, F, and H;X3012 is an amino acid selected from the group consisting of A, C, H, I,K, L, and R; X3013 is an amino acid selected from the group consistingof A, C, F, G, H, K, L, M, R, S, V, W, Y, and I; X3014 is an amino acidselected from the group consisting of A, C, F, G, H, I, L, M, N, Q, R,S, T, V, W, Y, and K; X3015 is an amino acid selected from the groupconsisting of A, K, and R; 3016 is an amino acid selected from the groupconsisting of A, F, K, and R; X3017 is an amino acid selected from thegroup consisting of A, C, F, G, I, K, L, N, Q, R, S, T, V, W, Y, H, A,and M; X3018 is an amino acid selected from the group consisting of A,C, F, I, K, L, M, Q, R, V, W, and Y; X3019 is an amino acid selectedfrom the group consisting of A, C, D, E, F, G, H, K, L, N, P, Q, R, V,W, Y, and I; X3020 is an amino acid selected from the group consistingof A, C, F, G, H, K, L, M, N, Q, R, V, W, Y, I, and P; and X3021 is anamino acid selected from the group consisting of A, C, H, I, K, L, M, N,P, Q, R, T, V, W, Y, F, and G.

In some aspects of the invention, the peptide comprises the sequence offormula (VIII), wherein X3001 is optionally an amino acid selected fromthe group consisting of A, C, D, G, I, K, L, M, N, P, Q, R, S, T, W, E,H, and Y, such as an amino acid selected from the group consisting of A,C, D, G, K, L, M, N, P, R, S, T, E, H, and Y (when X3001 is present);X3002 is optionally an amino selected from the group consisting of C, F,H, K, R, S, W, Y, G, I, and L, such as an amino acid selected from thegroup consisting of C, K, R, W, Y, G, I, and L (when X3002 is present);X3003 is optionally an amino acid selected from the group consisting ofA, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, and W, such as an aminoacid selected from the group consisting of A, C, G, H, I, K, L, M, R, S,T, and W; X3004 is optionally an amino acid selected from the groupconsisting of A, C, D, G, H, I, K, L, M, N, R, S, T, V, and P, such asan amino acid selected from the group consisting of A, C, G, H, I, K, L,M, N, R, S, T, and P; X3005 is optionally an amino acid selected fromthe group consisting of C, F, H, I, K, M, R, T, W, and Y, such as anamino acid selected from the group consisting of C, F, H, K, R, and W;X3006 is optionally an amino acid selected from the group consisting ofP, H, and A; X3007 is optionally an amino acid selected from the groupconsisting of C, G, R, W, A, and L, such as an amino acid selected fromthe group consisting of L, C, R, and W; X3008 is optionally an aminoacid selected from the group consisting of A, C, F, G, H, K, L, M, N, Q,R, T, V, W, Y, and I, such as an amino acid selected from the groupconsisting of A, C, F, H, K, R, V, W, Y, and I; X3009 is optionally anamino acid selected from the group consisting of C, I, R, V, and K, suchas an amino acid selected from the group consisting of C, R, V, and K;X3010 is optionally an amino acid selected from the group consisting ofA, C, G, H, I, K, L, M, Q, R, S, and T, such as an amino acid selectedfrom the group consisting of A, C, K, L, Q, R, and S; X3011 isoptionally an amino acid selected from the group consisting of A, I, K,L, M, R, S, V, W, C, F, and H, such as an amino acid selected from thegroup consisting of I, K, L, M, R, V, W, C, F, and H; X3012 isoptionally an amino acid selected from the group consisting of H and R(e.g., H); X3013 is optionally an amino acid selected from the groupconsisting of C, F, K, L, M, R, V, and I, such as an amino acid selectedfrom the group consisting of C, K, R, V, and I; X3014 is optionally anamino acid selected from the group consisting of A, M, C, F, H, I, L, N,R, S, V, W, and K, such as an amino acid selected from the groupconsisting of A, S, C, F, H, I, R, and K; X3015 is optionally K or R;X3016 is optionally K or R; X3017 is optionally an amino acid selectedfrom the group consisting of A, C, F, G, I, K, L, N, Q, R, S, T, V, W,H, A, and M, such as an amino acid selected from the group consisting ofC, G, I, K, L, N, Q, R, S, T, V, H, A, and M; X3018 is optionally anamino acid selected from the group consisting of A, K, C, I, L, R, and W(e.g., K, C, I, R, or W); X3019 is optionally an amino acid selectedfrom the group consisting of A, C, E, H, K, N, Q, R, and I, such as anamino acid selected from the group consisting of C, E, H, K, R, and I;X3020 is optionally an amino acid selected from the group consisting ofC, H, L, M, R, V, I, and P (e.g., C, M, I, or P); and X3021 isoptionally an amino acid selected from the group consisting of A, C, H,I, K, L, M, N, Q, R, V, W, Y, F, and G, such as an amino acid selectedfrom the group consisting of A, C, H, I, K, L, M, N, Q, R, V, W, F, andG.

The invention further provides a peptide that binds TFPI and comprisesat least amino acids 3-21 (X3003-X3021) of the structure of formula(IX):X3001-X3002-X3003-X3004-X3005-X3006-X3007-X3008-X3009-X3010-X3011-H-X3013-X3014-K/R-R-X3017-X3018-X3019-X3020-X3021(IX) (SEQ ID NO: 3121). In formula (IX), X3001 and X3002 areindependently either present or absent in the peptide. If present, X3001and/or X3002 are independently selected from any amino acid. Likewise,X3003, X3004, X3005, X3006, X3007, X3008, X3009, X3010, X3011, X3013,X3014, X3017, X3018, X3019, X3020 and X3021 are each independentlyselected from any amino acid. When present, X3001 is optionally an aminoacid selected from the group consisting of A, C, D, F, G, I, K, L, M, N,P, Q, R, S, T, W, E, H, and Y, such as an amino acid selected from thegroup consisting of A, C, D, G, I, K, L, M, N, P, Q, R, S, T, W, E, H,and Y (e.g., an amino acid selected from the group consisting of A, C,D, G, K, L, M, N, P, R, S, T, E, H, and Y). Likewise, when present,X3002 is optionally an amino acid selected from the group consisting ofA, C, D, F, H, K, M, N, P, R, S, T, W, Y, G, I, and L, such as an aminoacid selected from the group consisting of C, F, H, K, R, S, W, Y, G, I,and L (e.g., an amino acid selected from the group consisting of C, K,R, W, Y, G, I, and L). Also with respect to formula (IX), X3003 isoptionally an amino acid selected from the group consisting of A, C, D,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, and Y, such as an aminoacid selected from the group consisting of A, C, D, F, G, H, I, K, L, M,N, P, Q, R, S, T, and W (e.g., an amino acid selected from the groupconsisting of A, C, G, H, I, K, L, M, R, S, T, and W); X3004 isoptionally an amino acid selected from the group consisting of A, C, D,E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, and P, such as an aminoacid selected from the group consisting of A, C, D, G, H, I, K, L, M, N,R, S, T, V, and P (e.g., an amino acid selected from the groupconsisting of A, C, G, H, I, K, L, M, N, R, S, T, and P); X3005 isoptionally an amino acid selected from the group consisting of C, D, F,G, H, I, K, L, M, N, P, R, S, T, V, W, and Y, such as an amino acidselected from the group consisting of C, F, H, I, K, M, R, T, W, and Y(e.g., an amino acid selected from the group consisting of C, F, H, K,R, and W); X3006 is optionally an amino acid selected from the groupconsisting of A, W, C, K, P, R and H, such as an amino acid selectedfrom the group consisting of P, H, and A; X3007 is optionally an aminoacid selected from the group consisting of Q, A, C, F, G, H, I, K, L, N,R, S, T, W, and Y, such as an amino acid selected from the groupconsisting of C, G, R, W, A, and L (e.g., L, C, R, or W); X3008 isoptionally an amino acid selected from the group consisting of A, C, F,G, H, K, L, M, N, P, Q, R, S, T, V, W, Y, and I, such as an amino acidselected from the group consisting of A, C, F, G, H, K, L, M, N, Q, R,T, V, W, Y, and I (e.g., an amino acid selected from the groupconsisting of A, C, F, H, K, R, V, W, Y, and I); X3009 is optionally anamino acid selected from the group consisting of A, C, F, G, H, I, L, M,R, S, T, V, W, Y, and K, such as an amino acid selected from the groupconsisting of C, I, R, V, and K (e.g., C, R, V, or K); X3010 isoptionally an amino acid selected from the group consisting of A, C, F,G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, such as an amino acidselected from the group consisting of A, C, G, H, I, K, L, M, Q, R, S,and T (e.g., an amino acid selected from the group consisting of A, C,K, L, Q, R, and S); X3011 is optionally an amino acid selected from thegroup consisting of A, G, I, K, L, M, N, Q, R, S, T, V, W, Y, C, F, andH, such as an amino acid selected from the group consisting of A, I, K,L, M, R, S, V, W, C, F, and H (e.g., an amino acid selected from thegroup consisting of I, K, L, M, R, V, W, C, F, and H); X3013 isoptionally an amino acid selected from the group consisting of A, C, F,G, H, K, L, M, R, S, V, W, Y, and I, such as an amino acid selected fromthe group consisting of C, F, K, L, M, R, V, and I (e.g., C, K, R, V, orI); X3014 is optionally an amino acid selected from the group consistingof A, C, F, G, H, I, L, M, N, Q, R, S, T, V, W, Y, and K, such as anamino acid selected from the group consisting of A, M, C, F, H, I, L, N,R, S, V, W, and K (e.g., an amino acid selected from the groupconsisting of A, S, C, F, H, I, R, and K); X3017 is optionally an aminoacid selected from the group consisting of A, C, F, G, I, K, L, N, Q, R,S, T, V, W, Y, H, A, and M, such as an amino acid selected from thegroup consisting of A, C, F, G, I, K, L, N, Q, R, S, T, V, W, H, A, andM (e.g., an amino acid selected from the group consisting of C, G, I, K,L, N, Q, R, S, T, V, H, A, and M); X3018 is optionally an amino acidselected from the group consisting of A, C, F, I, K, L, M, Q, R, V, W,and Y, such as an amino acid selected from the group consisting of A, K,C, I, L, R, and W (e.g., K, C, I, R, or W); X3019 is optionally an aminoacid selected from the group consisting of A, C, D, E, F, G, H, K, L, N,P, Q, R, V, W, Y, and I, such as an amino acid selected from the groupconsisting of A, C, E, H, K, N, Q, R, and I (e.g., C, E, H, K, R, or I);X3020 is optionally an amino acid selected from the group consisting ofA, C, F, G, H, K, L, M, N, Q, R, V, W, Y, I, and P, such as an aminoacid selected from the group consisting of C, H, L, M, R, V, I, and P(e.g., C, M, I, or P); and/or X3021 is optionally an amino acid selectedfrom the group consisting of A, C, H, I, K, L, M, N, P, Q, R, T, V, W,Y, F, and G, such as an amino acid selected from the group consisting ofA, C, H, I, K, L, M, N, Q, R, V, W, Y, F, and G (e.g., an amino acidselected from the group consisting of A, C, H, I, K, L, M, N, Q, R, V,W, F, and G).

The TFPI-binding peptide of the invention comprises, in some aspects, anamino acid sequence having at least 60% identity (e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% or 100% identity) to the sequence of formula (X):Ac-GYASFPWFVQLHVHKRSWEMA-NH2 (X) (SEQ ID NO: 223). Optionally, thepeptide comprises or consists of the amino acid sequence of formula(VIII)-(IX) as defined herein. As used herein, “at least 60% identity”and similar terms encompass any integer from, e.g., 60%, to 100%, suchas 60%, 61%, 62%, and the like. Also, the term “at least [percentage]identity” encompasses any percentage that is greater than or equal tothe number of identical amino acids divided by the total number of aminoacids of the peptide of the invention ([at least percentageidentity]≧[number of identical amino acids]/[total number of amino acidsof the peptide of the invention]).

The invention also includes a peptide comprising or consisting of theamino acid sequence selected from the group consisting of SEQ ID NOs:2001-2498 (e.g., a peptide comprising or consisting of the amino acidsequence selected from the group consisting of SEQ ID NOs: 2001-2296 and2498 (such as SEQ ID NOs: 2001-2126, 2128-2296, or 2498) and/or selectedfrom the group consisting of SEQ ID NOs: 2297-2497 (such as SEQ ID NOs:2298-2497)). The invention further provides a peptide comprising orconsisting of the amino acid sequence selected from the group consistingof SEQ ID NOs: 3001-3108 (e.g., a peptide comprising or consisting ofthe amino acid sequence selected from the group consisting of SEQ IDNOs: 3001-3064 (such as SEQ ID NOs: 3001-3048, 3051-3053, 3055, or3057-3064) and/or selected from the group consisting of SEQ ID NOs:3065-3084 (such as SEQ ID NOs: 3066-3084) and/or selected from the groupconsisting of SEQ ID NOs: 3085-3108).

The peptide of SEQ ID NOs: 1-7 also, in some aspects, comprises one ormore amino acids attached at the N- or C-terminus of SEQ ID NOs: 1-7.For example, the invention includes a peptide comprising or consistingof the amino acid sequence of JBT0047, JBT0051, JBT0055, JBT0131,JBT0132, JBT0133, JBT0155, JBT0158, JBT0162, JBT0163, JBT0164, JBT0166,JBT0169, JBT0170, JBT0171, JBT0174, JBT0175, or JBT0293, all of whichcomprise the amino acid sequence of SEQ ID NO: 1. Exemplary peptidescomprising the amino acid sequence of SEQ ID NO: 2 include peptidescomprising or consisting of the amino acid sequence of JBT0294, JBT0295,JBT0296, JBT0297, JBT0298, JBT0299, JBT0300, JBT0301, JBT0302, JBT0303,JBT0304, JBT0305, JBT0306, JBT0307, JBT0308, JBT0309, JBT0310, orJBT0311. Exemplary peptides comprising the amino acid sequence of SEQ IDNO: 3 comprise or consist of the amino acid sequence of JBT0049,JBT0053, JBT0057, JBT0190, JBT0193, or JBT0197. The invention furtherincludes a peptide comprising or consisting of the amino acid sequenceof JBT0050, JBT0054, JBT0058, JBT0129, JBT0130, JBT0205, JBT0208,JBT0211, JBT0212, JBT0217, JBT0218, or JBT0219, all of which include theamino acid sequence of SEQ ID NO: 4. Exemplary peptides comprising SEQID NO: 5 include those comprising or consisting of the amino acidsequence of JBT0101, JBT0052, JBT0103, JBT0178, or JBT0182. Theinvention additionally includes a peptide comprising or consisting ofthe amino acid sequence of JBT0120, JBT0124, JBT0247, JBT0248, JBT0251,or JBT0252, each of which include the amino acid sequence of SEQ ID NO:6. A peptide including the amino acid sequence of SEQ ID NO: 7, e.g., apeptide comprising or consisting of the amino acid sequence of JBT0122,JBT0126. JBT0221, JBT0224, JBT0225, JBT0226, JBT0228, JBT0232, orJBT0233, also provided by the invention. The peptides described hereinare set forth in Table 5 of Example 1 and in FIGS. 12-18.

The invention further includes a TFPI-binding peptide comprising thestructure of formula (XI):X4001-Q-X4003-X4004-X4005-X4006-X4007-X4008-X4009-X4010-X4011-X4012-X4013-X4014-R-X4016-X4017-X4018-X4019-X4020(XI) (SEQ ID NO: 3151). With respect to formula (XI), X4001 is an aminoacid selected from the group consisting of F, L, M, Y, 1Ni, Thi, Bta,and Dopa (e.g., F, Y, 1Ni, Bta, or Dopa); X4003 is an amino acidselected from the group consisting of C, D, E, M, Q, R, S, T, Ede(O),and Cmc (e.g., D, E, or S); X4004 is an amino acid selected from thegroup consisting of Aib, E, G, I, K, L, M, P, R, W, and Y (e.g., K);X4005 is an amino acid selected from the group consisting of a, A, Aib,C, D, d, E, G, H, K, k, M, N, Nmg, p, Q, R, NpropylG, aze, pip, tic,oic, hyp, nma, Ncg, Abg, Apg, thz, and dtc (e.g., p, Nmg, NpropylG, aze,pip, tic, oic, or hyp); X4006 is an amino acid selected from the groupconsisting of A, C, C(NEM), D, E, G, H, K, M, N, Q, R, S, V, Cit,C(Acm), Nle, I, Ede(O), Cmc, Ed, Eea, Eec, Eef, Nif, and Eew (e.g., C,E, K, R, S, V, C(Acm), Nle, C(NEM), I, or Cit); X4007 is an amino acidselected from the group consisting of I, V, T, Chg, Phg, and Tle (e.g.,V or Tle); X4008 is an amino acid selected from the group consisting ofF, H, 1Ni, 2Ni, Pmy, and Y (e.g., H, 1Ni, 2Ni, or Pmy); X4009 is anamino acid selected from the group consisting of Aib, V, Chg, Phg, Abu,Cpg, Tle, and L-2-amino-4,4,4-trifluorobutyric acid (e.g., V, Abu, orTle); X4010 is an amino acid selected from the group consisting of A, C,D, d, E, F, H, K, M, N, P, Q, R, S, T, V, W, Y, Nmd, and C(NEM) (e.g.,D, P, C or T); X4011 is an amino acid selected from the group consistingof A, a, G, p, Sar, c, and hcy (e.g., G, a, c, hcy, or Sar); X4012 is anamino acid selected from the group consisting of Y, Tym, Pty, Dopa, andPmy (e.g., Y); X4013 is an amino acid selected from the group consistingof C, F, 1Ni, Thi, and Bta (e.g., F, 1Ni, or Bta); X4014 is an aminoacid selected from the group consisting of A, Aib, C, C(NEM), D, E, K,L, M, N, Q, R, T, V, and Hcy (e.g., Aib, C, E, or Hcy); X4016 is anamino acid selected from the group consisting of L, Hcy, Hle, and Aml;X4017 is an amino acid selected from the group consisting of A, a, Aib,C, c, Cha, Dab, Eag, Eew, H, Har, Hci, Hle, I, K, L, M, Nle, Nva, Opa,Orn, R, S, Deg, Ebc, Eca, Egz, Aic, Apc, and Egt (e.g., A, Aib, C, c,Aic, Eca, or Deg); X4018 is an amino acid selected from the groupconsisting of A, Aib, Hcy, hcy, C, c, L, Nle, M, N, and R (e.g., A, Aib,C, c, L, or Hcy); X4019 is an amino acid selected from the groupconsisting of K, R, and Har (e.g., K); and X4020 is an amino acidselected from the group consisting of K, L, Hcy, and Aml (e.g., L, Aml,and Hcy).

The TFPI-binding peptide of formula (XI) does not comprise the structureof formula (XII):X5001-Q-X5003-X5004-X5005-X5006-I/V-X5008-Aib/V-X5010-G-Y-X5013-X5014-R-L-X5017-X5018-K-K/L(XII) (SEQ ID NO: 3152). In formula (XII), X5001 is an amino acidselected from the group consisting of F, L, M, and Y; X5003 is an aminoacid selected from the group consisting of C, D, E, M, Q, R, S, and T;X5004 is an amino acid selected from the group consisting of E, G, I, K,L, M, P, R, W, and Y; X5005 is an amino acid selected from the groupconsisting of a, A, Aib, C, D, d, E, G, H, K, k, M, N, Nmg, Q, R, and p;X5006 is an amino acid selected from the group consisting of A, C, D, E,G, H, K, M, N, Q, R, S, and V; X5008 is an amino acid selected from thegroup consisting of F, H, and Y; X5010 is an amino acid selected fromthe group consisting of A, C, D, E, F, H, D, M, N, P, Q, R, S, T, V, W,and Y; X5013 is an amino acid selected from the group consisting of Aib,C, and F; X5014 is an amino acid selected from the group consisting ofA, Aib, C, D, E, K, L, M, N, Q, R, T, and V; X5017 is an amino acidselected from the group consisting of A, Aib, C, Cha, Dab, Eag, Eew, H,Har, Hci, Hle, I, K, L, M, Nle, Nve, Opa, Orn, R, and S; and X5018 is anamino acid selected from the group consisting of A, C, L, M, N, and R.

In one aspect, the TFPI-binding peptide described herein furthercomprises N-terminal amino acid(s) or moieties. For example, theTFPI-binding peptide of formula (XI) further comprises N-terminal aminoacid(s) and/or moieties linked to X4001, or X6001 and/or X7001 offormulas (XIII) and (XIV) (described below) are linked to N-terminalamino acid(s) or moieties. The N-terminal amino acid(s) and/or moietiesare optionally selected from the group consisting of FAM-Ttds, aproline-glutamate tag (“PE”), Palm, 2-phenyl acetyl, 3-phenyl propionyl,2-(naphth-2-yl) acetyl, hexanoyl, 2-methyl propionyl, 3-methyl butanoyl,2-naphthylsulfonyl, acetyl, Con, Con(Meox), AOA, Oxme-AOA, Meox-Lev,levulinic acid (Lev), and pentynoic acid (Pyn), and 1-naphthylsulfonyl.Alternatively or in addition, the TFPI-binding peptide (e.g., theTFPI-binding peptide of formula (XI), formula (XIII), and/or formula(XIV)) further comprises one or more amino acid(s) and/or moietieslinked to the C-terminal amino acid (e.g., X4020, X6020, or X7022 orX7023). The C-terminal amino acid(s) and/or moieties are optionallyselected from the group consisting of C, c, C(NEM),K(Ttds-maleimidopropionyl(EtSH)), FA19205, FA19204, FA19203, FA03202,K(Tdts-maleimid), K(AOA), Cea, and amide. Alternatively, the C-terminalamino acid(s) and/or moieties also are selected from the groupconsisting of Eag, Con, Con(Meox), Hly, K, Orn, Dab, Dap, Hcy, Pen,K(Myr), K(Ttds-Myr), K(Ttds-Palm), K(Ttds-Ac), K(Ttds-γGlu-Myr),K(AlbuTag), and K(4PBSA). In the context of formula (XI), C-terminalamino acid(s) and/or moieties are designated herein as X4021. In thecontext of formulas (XIII) and (XIV), C-terminal amino acid(s) and/ormoieties are designated as X6021 and X7024, respectively.

In one embodiment, the peptide comprises a cyclic structure formedbetween X4018 and X4021. In this regard, X4018 is optionally C or c, andX4021 is optionally Cea. In another embodiment, the peptide comprises acyclic structure formed between X4011 and X4014. In this regard, X4011is optionally c or hcy, and X4014 is optionally C or Hcy.

The invention also includes a peptide consisting of the amino acidsequence selected from the group consisting of SEQ ID NOs: 4022, 4024,4032, 4036-4047, 4049-4078, 4086-4097, 4100-4127, 4129-4170, 4173-4195,4200-4214, 4217-4225, 4228, 4230, 4231, 4238, and 4239, as well as apeptide consisting of the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1294-1336, 4002, 4013, 4021, 4023, 4025-4031,4033-4035, 4048, 4079-4085, 4098, 4099, 4128, 4171, 4172, 4196-4199,4215, 4216, 4226, 4277, 4229, 4232, and 4233.

In certain embodiments, the peptide of the invention comprises orconsists of the amino acid sequence of JBT0047, JBT0049, JBT0101,JBT0120, or JBT0122 or any of the inventive peptides described herein(e.g., a peptide comprising or consisting of the amino acid sequence ofany one of SEQ ID NOs: 1-3108, such as a peptide comprising orconsisting of the amino acid sequence of any one of SEQ ID NOs: 8-741,744-968, 971-978, 1001-1210, 1213-1289, 1290-1293, 2001-2126, 2128-2296,2298-2498, 3001-3048, 3051-3053, 3055, 3057-3064, and 3067-3108; apeptide comprising or consisting of the amino acid sequence of any oneof SEQ ID NOs: 4022, 4024, 4032, 4036-4047, 4049-4078, 4086-4097,4100-4127, 4129-4170, 4173-4195, 4200-4214, 4217-4225, 4228, 4230, 4231,4238, and 4239; or a peptide comprising or consisting of the amino acidsequence selected from the group consisting of SEQ ID NOs: 1294-1336,4002, 4013, 4021, 4023, 4025-4031, 4033-4035, 4048, 4079-4085, 4098,4099, 4128, 4171, 4172, 4196-4199, 4215, 4216, 4226, 4277, 4229, 4232,and 4233), or a peptide comprising or consisting of the amino acidsequence selected from the group consisting of SEQ ID NOs: 1337-1355,3146-3154 and 4240-4268, or a variant of any of the foregoing. By“variant” is meant a peptide comprising one or more amino acidsubstitutions, amino acid deletions, or amino acid additions to a parentamino acid sequence. Variants include, but are not limited to, peptideshaving an amino acid sequence that is at least 60%, 65%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to any of the amino acid sequences provided herein whileretaining the ability to bind TFPI and/or inhibit TFPI activity. In oneembodiment, the peptide comprises or consists of the amino acid sequenceof JBT0132, JBT0303, JBT0193, JBT0178, JBT0120, or JBT0224.

In one aspect, the peptide of the invention consists of 40 amino acidsor less, such as 35 amino acids or less. Optionally, the peptide of theinvention consists of 25 amino acids or less, or 10 amino acids or less.In various embodiments, the peptide comprises 15-35 amino acid residues(e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, or 35 amino acid residues). However, it is alsocontemplated that a peptide described herein comprising one or moredeletions is suitable in the context of the invention so long as thepeptide binds TFPI and, optionally, blocks TFPI inhibition of thecoagulation cascade. In some aspects, amino acids are removed fromwithin the amino acid sequence, at the N-terminus, and/or at theC-terminus. Such peptide fragments can comprise 3-14 amino acid residues(e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acid residues).

Optionally, the peptide of the invention comprises one or more aminoacid substitutions (with reference to any of the amino acid sequencesprovided herein) that do not destroy the ability of the peptide to bindand/or inhibit TFPI. For instance, peptides comprising or consisting ofthe amino acid sequence selected from the group consisting of JBT0294,JBT0295, JBT0296, JBT0297, JBT0298, JBT0299, JBT0300, JBT0301, JBT0302,JBT0303, JBT0304, JBT0305, JBT0306, JBT0307, JBT0308, JBT0309, JBT0310,or JBT0311 are substitutional mutants of the amino acid sequence ofJBT0293 (the amino acid sequence of SEQ ID NO: 1 directly linked to aphenylalanine residue at the N-terminus and a lysine reside at theC-terminus) (see FIG. 4).

Amino acid substitutions include, but are not limited to, those which:(1) reduce susceptibility to proteolysis, (2) reduce susceptibility tooxidation, (3) alter binding affinities, and/or (4) confer or modifyother physiochemical or functional properties on a peptide. In oneaspect, the substitution is a conservative substitution, wherein anamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined within the art, and include amino acids withbasic side chains (e.g., lysine, arginine, and histidine), acidic sidechains (e.g., aspartic acid and glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, andtryptophan), beta-branched side chains (e.g., threonine, valine, andisoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, and histidine). It will be appreciated, however, that apractitioner is not limited to creating conservative substitutions solong as the resulting peptide retains the ability to downregulate, inwhole or in part, TFPI activity. The invention also embracesTFPI-binding peptides (e.g., TFPI-inhibitory peptides) comprisingatypical, non-naturally occurring amino acids, which are well known inthe art. Exemplary non-naturally occurring amino acids includeornithine, citrulline, hydroxyproline, homoserine, phenylglycine,taurine, iodotyrosine, 2,4-diaminobutyric acid, α-amino isobutyric acid,4-aminobutyric acid, 2-amino butyric acid, γ-amino butyric acid, 2-aminoisobutyric acid, 3-amino propionic acid, norleucine, norvaline,sarcosine, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, β-alanine, a fluoro-amino acid, a3-methyl amino acid, β-C-methyl amino acid, a N-methyl amino acid,2-amino-isobutyric acid, β-homoglutamatic acid, β-homophenylalanine,β-homolysine, β-homoleucine, β-homoasparagine, β-homoglutamine,β-homoarginine, β-homoserine, β-homotyrosine, β-homoaspartic acid,β-homovaline, β-homoasparagin, (S)-cyclohexylalanine, (S)-citrullin,(S)-2,4-diaminobutyric acid, (S)-2,4-diaminobutyric acid,(S)-diaminopropionic acid, (S)-2-propargylglycine,(S)—N(omega)-nitro-arginine, L-homophenylalanine, S)-homo-arginine,(S)-homo-citrulline, (S)-homo-cysteine, (S)-2-amino-5-methyl-hexanoicacid, (S)-homo-lysine, (S)-norleucine, (S)—N-methylalanine,(S)—N-methyl-aspartic acid, (S)—N-methyl-glutamic acid,(S)—N-methyl-phenylalanine, N-methyl-glycine, (S)—N-methyl-lysine,(S)—N-methyl-leucine, (S)—N-methyl-arginine, (S)—N-methyl-serine,(S)—N-methyl-valine, (S)—N-methyl-tyrosine, (S)-2-amino-pentanoic acid,(S)-2-pyridyl-alanine, (S)-ornithine, L-phenylglycin, 4-phenyl-butyricacid and selenomethionine. The individual amino acids may have either Lor D stereochemistry when appropriate, although the L stereochemistry istypically employed for all of the amino acids in the peptide.

The invention further includes TFPI-binding peptide (e.g.,TFPI-inhibitory peptide) variants comprising one or more amino acidsinserted within an amino acid sequence provided herein and/or attachedto the N-terminus or C-terminus. In one aspect, the peptide furthercomprises one or more amino acids that facilitate synthesis, handling,or use of the peptide, including, but not limited to, one or two lysinesat the N-terminus and/or C-terminus to increase solubility of thepeptide. Suitable fusion proteins include, but are not limited to,proteins comprising a TFPI-binding peptide (e.g., TFPI-inhibitorypeptide) linked to one or more polypeptides, polypeptide fragments, oramino acids not generally recognized to be part of the protein sequence.In one aspect, a fusion peptide comprises the entire amino acidsequences of two or more peptides or, alternatively, comprises portions(fragments) of two or more peptides. In addition to all or part of theTFPI-binding peptides (e.g., TFPI-inhibitory peptides) described herein,a fusion protein optionally includes all or part of any suitable peptidecomprising a desired biological activity/function. Indeed, in someaspects, a TFPI-binding peptide (e.g., TFPI-inhibitory peptide) isoperably linked to, for instance, one or more of the following: apeptide with long circulating half life, a marker protein, a peptidethat facilitates purification of the TFPI-binding peptide (e.g.,TFPI-inhibitory peptide), a peptide sequence that promotes formation ofmultimeric proteins, or a fragment of any of the foregoing. Suitablefusion partners include, but are not limited to, a His tag, a FLAG tag,a strep tag, and a myc tag. Optionally, the TFPI-binding peptide (e.g.,TFPI-inhibitory peptide) is fused to one or more entities that enhancethe half life of the peptide. Half life can be increased by, e.g.,increasing the molecular weight of the TFPI-binding peptide to avoidrenal clearance and/or incorporating a ligand for the nFcreceptor-mediated recycling pathway. In one embodiment, the TFPI-bindingpeptide is fused to or chemically conjugated to (as described furtherbelow) an albumin polypeptide or a fragment thereof (e.g., human serumalbumin (HSA) or bovine serum albumin (BSA)). The albumin fragmentcomprises 10%, 25%, 50%, or 75% of the full length albumin protein.Alternatively or in addition, the TFPI-binding peptide comprises analbumin binding domain or fatty acid that binds albumin whenadministered in vivo. An example of an albumin binding domain is“albu-tag,” a moiety derived from on 4-(p-iodophenyl)-butanoic acid(Dumelin et al., Angew Chem Int Ed Engl 47:3196-3201 (2008)). Othersuitable fusion partners include, but are not limited to, aproline-alanine-serine multimer (PASylation) and an antibody or fragmentthereof (e.g., an Fc portion of an antibody).

In one embodiment, two or more TFPI-binding peptides are fused together,linked by a multimerization domain, or attached via chemical linkage togenerate a TFPI-binding peptide (e.g., TFPI-inhibitory peptide) complex.The TFPI-binding peptides may be the same or different. Thus, theinvention provides a homo-dimer (i.e., a dimer comprising two identicalTFPI-binding peptides), a homo-multimer (i.e., a complex comprisingthree or more identical TFPI-binding peptides), a hetero-dimer (i.e., adimer comprising two different TFPI-binding peptides), andhetero-multimer (i.e., a complex comprising three or more TFPI-bindingpeptides, wherein at least two of the TFPI-binding peptides aredifferent) comprising or consisting of any of the peptides describedherein, optionally attached by one or more linkers.

In this regard, the invention provides a peptide complex comprising afirst peptide and a second peptide. Any of the peptides described hereinare suitable subunits (e.g., first peptide or second peptide) for thepeptide complex. In some embodiments, the peptide complex comprises25-100 amino acids, e.g., 30-80 amino acids, 30-60 amino acids, or 30-50amino acids. The first peptide and the second peptide may bind differentregions (epitopes) of human TFPI and, optionally, the peptide complexinhibits two or more TFPI functions, such as, but not limited to, TFPIbinding to FXa and TFPI binding to FVIIa. The level of inhibition of atleast one TFPI (e.g., TFPI-1) activity mediated by the peptide complexis greater than the level of inhibition achieved by the first peptide orthe second peptide, alone or in combination, in various embodiments ofthe invention. The different regions (epitopes) on TFPI may be on thesame TFPI polypeptide, or on different TFPI polypeptides. The functionalcharacteristics and therapeutic and diagnostic applications of monomericTFPI-binding peptides described herein also are applicable to thepeptide complexes described herein. Similarly, descriptions ofmodifications to monomeric TFPI-binding peptides also relate to peptidecomplexes.

The invention provides a peptide complex comprising a first peptidecomprising the structure of formula (XIII):X6001-X6002-X6003-X6004-X6005-X6006-X6007-X6008-X6009-X6010-X6011-X6012-X6013-X6014-X6015-X6016-X6017-X6018-X6019-X6020(XIII) (SEQ ID NO: 3153). In formula (XIII),

X6001 is an amino acid selected from the group consisting of F, L, M, Y,1Ni, Thi, Bta, Dopa, Bhf, C, D, G, H, I, K, N, Nmf, Q, R, T, V, and W,such as an amino acid selected from the group consisting of 1Ni, Bta,Dopa, F, L, Y and M;

X6002 is an amino acid selected from the group consisting of Q, G, andK, such as Q;

X6003 is an amino acid selected from the group consisting of C, D, E, M,Q, R, S, T, Ede(O), Cmc, A, Aib, Bhs, F, G, H, I, K, L, N, P, V, W andY, such as an amino acid selected from the group consisting of D, E, S,M, Q, R, T and C;

X6004 is an amino acid selected from the group consisting of Aib, E, G,I, K, L, M, P, R, W, Y, A, Bhk, C, D, F, H, k, N, Nmk, Q, S, T, and V,such as an amino acid selected from the group consisting of K, Aib, L,P, R, E, G, I, Y, M and W;

X6005 is an amino acid selected from the group consisting of a, A, Aib,C, D, d, E, G, H, K, k, M, N, Nmg, p, Q, R, NpropylG, aze, pip, tic,oic, hyp, nma, Ncg, Abg, Apg, thz, dtc, Bal, F, L, S, T, V, W and Y,such as an amino acid selected from the group consisting of p, Nmg,NpropylG, aze, pip, tic, oic, hyp, a, Aib, D, d, G, H, K, k, N, Q, R, A,E, C and M;

X6006 is an amino acid selected from the group consisting of A, C,C(NEM), D, E, G, H, K, M, N, Q, R, S, V, Cit, C(Acm), Nle, I, Ede(O),Cmc, Ed, Eea, Eec, Eef, Nif, Eew, Aib, Btq, F, I, L, T, W and Y, such asan amino acid selected from the group consisting of C, E, K, R, S, V,C(Acm), Nle, C(NEM), I, Cit, A, D, G, H, N, Q and M;

X6007 is an amino acid selected from the group consisting of I, V, T,Chg, Phg, Tle, A, F, G, I, K, L, Nmv, P, Q, S, W and Y, such as Tle, Vor I;

X6008 is an amino acid selected from the group consisting of F, H, 1Ni,2Ni, Pmy, Y, and W, such as an amino acid selected from the groupconsisting of H, 1Ni, 2Ni, Pmy, F and Y;

X6009 is an amino acid selected from the group consisting of Aib, V,Chg, Phg, Abu, Cpg, Tle, L-2-amino-4,4,4-trifluorobutyric acid, A, f, I,K, S, T and V, such as V, Abu or Tle;

X6010 is an amino acid selected from the group consisting of A, C, D, d,E, F, H, K, M, N, P, Q, R, S, T, V, W, Y, Nmd, C(NEM), Aib, G, I, L andNmf, such as an amino acid selected from the group consisting of D, P,C, T, A, E, K, M, N, Q, R, F, H, S, V, W and Y;

X6011 is an amino acid selected from the group consisting of A, a, G, p,Sar, c, hcy, Aib, C, K, G and Nmg, such as G, a, c, hcy or Sar;

X6012 is an amino acid selected from the group consisting of Y, Tym,Pty, Dopa, and Pmy, such as Y;

X6013 is an amino acid selected from the group consisting of Aib, C, F,1Ni, Thi, Bta, A, E, G, H, K, L, M, Q, R, W and Y, such as an amino acidselected from the group consisting of F, 1Ni, Bta and C;

X6014 is an amino acid selected from the group consisting of A, Aib, C,C(NEM), D, E, K, L, M, N, Q, R, T, V, Hcy, Bhe, F, G, H, I, P, S, W andY, such as an amino acid selected from the group consisting of Aib, C,E, Hcy, A, D, K, L, M, N, Q, R, T, V and Aib;

X6015 is an amino acid selected from the group consisting of R,(omega-methyl)-R, D, E and K, such as R;

X6016 is an amino acid selected from the group consisting of L, Hcy,Hle, and Aml, such as an amino acid selected from the group consistingof L, Aml, Hle and Hcy;

X6017 is an amino acid selected from the group consisting of A, a, Aib,C, c, Cha, Dab, Eag, Eew, H, Har, Hci, Hle, I, K, L, M, Nle, Nva, Opa,Orn, R, S, Deg, Ebc, Eca, Egz, Aic, Apc, Egt, (omega-methyl)-R, Bhr,Cit, D, Dap, E, F, G, N, Q, T, V, W and Y, such as an amino acidselected from the group consisting of A, Aib, C, c, Aic, Eca, Deg, Cha,Dab, Dap, Eag, Eew, H, Har, Hci, Hle, K, Nle, Nva, Opa, Orn, R, I, L, Sand M;

X6018 is an amino acid selected from the group consisting of A, Aib,Hcy, hcy, C, c, L, Nle, M, N, R, Bal, D, E, F, G, H, I, K, Q, S, T, V, Wand Y, such as an amino acid selected from the group consisting of A,Aib, C, c, L, Hcy, N, M and R;

X6019 is an amino acid selected from the group consisting of K, R, Har,Bhk and V, such as K; and

X6020 is an amino acid selected from the group consisting of K, L, Hcy,Aml, Aib, Bhl, C, F, G, H, I, Nml, Q, R, S, T, V, W and Y, such as anamino acid selected from the group consisting of L, Aml, Hcy and K.

Alternatively, the first peptide comprises the structure of any one offormulas (I)-(IV) and (XI) described herein. The first peptide, invarious embodiments, comprises (or consists of) an amino acid sequenceselected from the group consisting of SEQ ID NOs: 8-978, 4022, 4024,4032, 4036-4047, 4049-4078, 4086-4097, 4100-4127, 4129-4170, 4173-4195,4200-4214, 4217-4225, 4228, 4230, 4231, 4238, 4239, 4002, 4013, 4021,4023, 4025-4031, 4033-4035, 4048, 4079-4085, 4098, 4099, 4128, 4171,4172, 4196-4199, 4215, 4216, 4226, 4277, 4229, 4232, and 4233.

The second peptide, in one aspect, comprises the structure of formula(XIV): X7001-X7002-X7003-X7004-X7005-X7006-(XIV) (SEQ ID NO: 3154). Informula (XIV),

X7001 is either present or absent, whereby in case X7001 is present itis an amino acid selected from the group consisting of A, C, C(NEM), D,E, F, G, H, I, K, L, P, R, S, T, V and W, such as an amino acid selectedfrom the group consisting of A, D, F, G, H, K, L and S;

X7002 is either present or absent, whereby in case X7002 is present itis an amino acid selected from the group consisting of A, C, C(NEM), D,E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y, such as an aminoacid selected from the group consisting of H, F, M and R;

X7003 is an amino acid selected from the group consisting of A, F, I, K,L, R, S, T, V, W and Y, such as F or Y;

X7004 is an amino acid selected from the group consisting of A, D, E, F,G, I, K, L, R, S, T, V and W, such as K;

X7005 is R or W, such as W;

X7006 is an amino acid selected from the group consisting of F, H, I, K,L, R, V and W, such as F or H;

X7007 is an amino acid selected from the group consisting of Orn, homoK,C, Hcy, Dap and K, such as C or Hcy;

X7008 is an amino acid selected from the group consisting of A, G, R, Sand T, such as A, G or S;

X7009 is an amino acid selected from the group consisting of a, A, I, K,L, M, m, Moo, Nle, p, R, Sem and V, such as M, Sem, or V;

X7010 is an amino acid selected from the group consisting of A, G, I, K,L, P, R, S, T and V, such as K, P or R;

X7011 is an amino acid selected from the group consisting of D, E, G, Sand T, such as D;

X7012 is an amino acid selected from the group consisting of A, a, D, d,E, e, F, f, G, I, K, k, L, l, M, m, Moo, Nle, nle, P, p, R, r, S, s,Sem, T, t, V, v, W and w, such as an amino acid selected from the groupconsisting of F, L, l, M and Sem;

X7013 is an amino acid selected from the group consisting of A, C,C(NEM), Con, Con(Meox), D, d, E, e, Eag, F, G, I, K, L, N, R, S, s, T, Vand W, such as an amino acid selected from the group consisting of D, G,K and S;

X7014 is an amino acid selected from the group consisting of A, D, E, F,G, I, K, L, M, R, S, T, V and W, such as G;

X7015 is an amino acid selected from the group consisting of A, D, E, F,G, I, K, L, M, Nle, R, S, T, V and W, such as I or T;

X7016 is an amino acid selected from the group consisting of A, D, E, F,I, K, L, M, Moo, Nle, R, S, Sem, T, V, W and Y, such as an amino acidselected from the group consisting of D, F, M, Sem and Y;

X7017 is an amino acid selected from the group consisting of A, D, E, F,G, I, K, L, R, S, T, V, W and Y, such as S or T;

X7018 is an amino acid selected from the group consisting of C and D,such as C;

X7019 is an amino acid selected from the group consisting of A, F, I, L,S, T, V and W, such as A or V;

X7020 is an amino acid selected from the group consisting of F and W,such as W;

X7021 is an amino acid selected from the group consisting of I, L and V,such as V;

X7022 is an amino acid selected from the group consisting of A, D, E, F,G, I, K, L, P, R, S, T, V and W, such as the group consisting of F, L,K, R, P and W;

X7023 is either present or absent, whereby in case X7023 is present itis an amino acid selected from the group consisting of A, C, C(NEM),Con, Con(Meox), D, E, Eag, F, G, I, K, L, R, S, T, V, W and Y, such asan amino acid selected from the group consisting of A, D, F, M, S and Y;and the peptide comprises as a cyclic structure generated by a linkagebetween X7007 and X7018.

Alternatively, the second peptide comprises the structure of any one offormulas (V)-(VII) described herein. The second peptide, in someembodiments, comprises (or consists of) an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1001-1336. While peptides offormula (XIII) and formula (XIV) are referenced herein as first andsecond peptides of a peptide complex, monomeric peptides having thestructure of formula (XIII) or formula (XIV) also are contemplated.

Examples of TFPI-binding peptides that bind different TFPI epitopesinclude JBT1857, an example of the JBT0047 class of peptides(represented as, e.g., formulas (I)-(IV) and (XI) and examples of whichare set forth in FIGS. 32, 62, and 65), and JBT1837, an example of theJBT0120 class of peptides (represented as, e.g., formulas (V)-(VII) andexamples of which are set forth in FIG. 34). Thus, in one aspect, theinvention provides a peptide complex comprising a peptide subunit (e.g.,a first peptide) comprising an amino acid sequence at least 80%, atleast 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:178 (JBT1857) or SEQ ID NO: 4261 (JBT2548). Additionally, the inventionprovides a peptide complex comprising a peptide subunit (e.g., a secondpeptide) comprising an amino acid sequence at least 80%, at least 85%,at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1044(JBT1837). Exemplary TFPI-binding peptide dimers include JBT2496 (SEQ IDNO: 4211), JBT2547 (SEQ ID NO: 4260), JBT2521 (SEQ ID NO: 4242), JBT2522(SEQ ID NO: 4243), JBT2523 (SEQ ID NO: 4244), and JBT2533 (SEQ ID NO:4250).

In various aspects of the disclosure, the peptide subunits (e.g., firstpeptide and second peptide) of the peptide complex are fused togetherdirectly or are linked by a linker moiety. For example, the firstpeptide and the second peptide are conjugated together by reacting anucleophilic reactive moiety on one peptide with an electrophilicreactive moiety on another peptide or by oxidation to form a disulfide.In exemplary embodiments, the first and second peptides are linked by anamide bond, such as an amide bond that forms upon reaction of an amineon one peptide with a carboxyl group on another peptide. It will beappreciated that peptides are optionally derivatized with a derivatizingagent before conjugation.

In some embodiments, the first peptide and the second peptide (andoptionally additional peptides) are linked by a linker moiety. Anylinker moiety is suitable for use in the context of the peptide complex.The linker moiety, in some aspects of the invention, bridges a distanceof about 1 Å to about 100 Å, e.g., about 5 Å to about 80 Å (about 5 Å toabout 50 Å), about 10 Å to about 70 Å (about 10 Å to about 60 Å, about10 Å to about 50 Å, about 10 Å to about 40 Å, or about 10 Å to about 30Å), in one of its conformations. Thus, the linker is optionally about 1Å to about 100 Å in length, e.g., about 5 Å to about 50 Å or about 10 Åto about 30 Å in length in one of its conformations. Linkers of greaterlength (greater than about 100 Å) also are contemplated. For example,biocompatible polymers, optionally having a molecular weight of about 2kDa to about 60 kDa, also are contemplated for use in the peptidecomplex. Examples of biocompatible polymers include, but are not limitedto, PEG, PSA, proline-alanine-serine multimer, and hydroxyethyl starch.

In one non-limiting example, the linker moiety is a molecule with atleast two reactive groups (before conjugation to the first and secondpeptides) capable of reacting with each of the first peptide and thesecond peptide. In some embodiments, the linker moiety has only tworeactive groups and is bifunctional. The reactive groups are bothnucleophilic, both electrophilic, or a combination of nucleophilic andelectrophilic reactive groups. Nonlimiting combinations of reactivegroups are shown in FIG. 73. The linker moiety may contain structuralelements resulting from a chemical ligation, such as, for instance,cystein, oxime, hydrazide, succinimide, thioether, triazole, secondaryamine, amide, disulfide. In various embodiments, the linker moiety isattached to the first peptide and/or the second peptide via an oxime, ahydrazide, a succinimide, a thioether, a triazole, a secondary amine, anamide, or a disulfide. Additional description of linker moieties andreactive groups is provided in International Patent Publication No. WO2011/143209, incorporated herein by reference in its entirety.

Hydrophobic linkers also are suitable for use in the context of theinvention. Hydrophobic linkers are known in the art. See, e.g.,Bioconjugate Techniques, G. T. Hermanson (Academic Press, San Diego,Calif., 1996), which is incorporated by reference in its entirety.Suitable hydrophobic linker moieties include, for example,8-hydroxyoctanoic acid and 8-mercaptooctanoic acid. Hydrophilic linkermoieties such as, for example, polyalkylene glycol, also are suitablefor use in the context of the invention. In some embodiments, the linkermoiety comprises a chain of atoms from about 1 to about 60 atoms long.In some embodiments, the chain atoms are all carbon atoms. In someembodiments, the chain atoms in the backbone of the linker are selectedfrom the group consisting of C, O, N, and S. Chain atoms and linkers maybe selected according to their expected solubility (hydrophilicity) soas to provide a more soluble peptide complex.

In one aspect, the linker moiety comprises the structure Z₁₋₂₀, whereinZ is an oligomer building block. Examples of oligomer building blocksinclude, but are not limited to, an amino acid, hydroxy acid, ethyleneglycol, propylene glycol, or a combination of any of the foregoing. Forexample, the linker moiety is optionally an amino acid, a dipeptide, atripeptide, or a polypeptide comprising 4-20 amino acids. In someembodiments, Z is G, s, S, a, A, Bal, Gaba, Ahx, Ttds, or a combinationof any of the foregoing (such as peptide ten-mer comprising A, S, or acombination of A and S). If desired, the linking moiety comprises anamine, ether, thioether, maleimide, disulfide, amide, ester, alkene,cycloalkene, alkyne, trizoyl, carbamate, carbonate, cathepsinB-cleavable, or hydrazone.

The terms “first peptide” and “second peptide” are not meant to imply aparticular physical order of the peptides, but merely to distinguishdifferent subunits of the peptide complex. The subunits of the peptidecomplex may be linked in any of a number of configurations so long asthe first peptide and second peptide interact with TFPI. For example,the C-terminus of the first peptide is connected to the N-terminus ofthe second peptide, the N-terminus of the first peptide is connected tothe C-terminus of the second peptide, the N- or C-terminus of the first(or second) peptide is connected to an internal attachment point in thesecond (or first) peptide, or the first and second peptides areconnected via internal attachment points (i.e., attachment pointslocated within the amino acid sequence of the peptide and not at the N-or C-terminus). Exemplary attachment points for a linking moiety in thefirst peptide of formula (XIII) is the N-terminal amino group, theC-terminal carboxylic acid, X6004, X6006, X60010, or X60014 via anappropriate functional group in the amino acid side chain, such as, butnot limited to, amine, carboxylic acid, thiol, alkene, alkyne, azide,carbonyl, aminooxy, hydrazine and halogens. More than one linker may beused, e.g., a first linking moiety is attached at the N-terminus of thefirst peptide and the C-terminus of the second peptide, and a secondlinking moiety (which may be the same type of moiety or a different typeof moiety) is attached at the C-terminus of the first peptide andattached at the N-terminus of the second peptide. While the discussionof possible configurations refers to the first and second peptides, itwill be appreciated that additional peptides may be linked to the firstand/or second peptides as described herein.

“Derivatives” are included in the invention and include TFPI-bindingpeptides that have been chemically modified in some manner distinct fromaddition, deletion, or substitution of amino acids. In this regard, apeptide of the invention provided herein is chemically bonded withpolymers, lipids, other organic moieties, and/or inorganic moieties.Examples of peptide and protein modifications are given in Hermanson,Bioconjugate Techniques, Academic Press, (1996). The TFPI-bindingpeptides described herein optionally comprise a functional group thatfacilitates conjugation to another moiety (e.g., a peptide moiety).Exemplary functional groups include, but are not limited to,isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride,aldehyde, epoxide, oxirane, carbonate, arylating agent, imidoester,carbodiimide, anhydride, alkyl halide derivatives (e.g., haloacetylderivatives), maleimide, aziridine, acryloyl derivatives, arylatingagents, thiol-disulfide exchange reagents (e.g., pyridyl disulfides orTNB thiol), diazoalkane, carboyldiimadazole, N,N′-Disuccinyl carbonate,N-Hydroxysuccinimidyl chloroformate, and hydrazine derivatives.Maleimide is useful, for example, for generating a TFPI-binding peptidethat binds with albumin in vivo.

Derivatives are prepared in some situations to increase solubility,stability, absorption, or circulating half life. Various chemicalmodifications eliminate or attenuate any undesirable side effect of theagent. In one aspect, the invention includes TFPI-binding peptidescovalently modified to include one or more water soluble polymerattachments. A water soluble polymer (or other chemical moiety) isattached to any amino acid residue, although attachment to the N- orC-terminus is preferred in some embodiments. Optionally, a polymer isattached to the peptide via one or more amino acids or building blocksthat offer functional groups that facilitate polymer attachment. Forexample, JBT2315 comprises a C-terminal cysteine (position X4021 withrespect to formula (XI)), which facilitates the addition of, e.g., amaleimide polyethylene glycol (PEG). Useful polymers include, but arenot limited to, PEG (e.g., PEG approximately 40 kD, 30 kD, 20 kD, 10,kD, 5 kD, or 1 kD in size), polyoxyethylene glycol, polypropyleneglycol, monomethoxy-polyethylene glycol, dextran, hydroxyethyl starch,cellulose, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propyleneglycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer,polysialic acid (PSA), polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol, as well as mixtures of any of the foregoing. In oneaspect, the peptide of the invention is a PEGylated peptide. PEGmoieties are available in different shapes, e.g., linear or branched.For further discussion of water soluble polymer attachments, see U.S.Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and4,179,337. Other moieties useful for improving peptide half life orstability are described herein and include, for instance, albumin(optionally modified to allow conjugation to the inventive peptide),fatty acid chains (e.g., C12-C18 fatty acid, such as a C14 fatty acid),an antibody or fragment thereof (e.g., an Fc portion of an antibody),and proline-alanine-serine multimers.

In another aspect, a peptide derivative includes a targeting moietyspecific for a particular cell type, tissue, and/or organ.Alternatively, the peptide is linked to one or more chemical moietiesthat facilitate purification, detection, multimerization, binding withan interaction partner, and characterization of peptide activity. Anexemplary chemical moiety is biotin. Other moieties suitable forconjugation to the TFPI-binding peptide of the invention include, butare not limited to, a photosensitizer, a dye, a fluorescence dye, aradionuclide, a radionuclide-containing complex, an enzyme, a toxin, anda cytotoxic agent. Photosensitizers include, e.g., Photofrin, Visudyne,Levulan, Foscan, Metvix, Hexvix®, Cysview™, Laserphyrin, Antrin,Photochlor, Photosens, Photrex, Lumacan, Cevira, Visonac, BF-200 ALA,and Amphinex. If desired, a His tag, a FLAG tag, a strep tag, or a myctag is conjugated to the peptide.

In addition, in one aspect, the peptides of the invention are acylatedat the N-terminal amino acid of the peptide. In another aspect, thepeptides of the invention are amidated at the C-terminal amino acid ofthe peptide. In a still further aspect, the peptides of the inventionare acylated at the N-terminal amino acid of the peptide and areamidated at the C-terminal amino acid of the peptide.

Derivatives also include peptides comprising modified ornon-proteinogenic amino acids or a modified linker group (see, e.g.,Grant, Synthetic Peptides: A User's Guide, Oxford University Press(1992)). Modified amino acids include, for example, amino acids whereinthe amino and/or carboxyl group is replaced by another group.Non-limiting examples include modified amino acids incorporatingthioamides, ureas, thioureas, acylhydrazides, esters, olefines,sulfonamides, phosphoric acid amides, ketones, alcohols, boronic acidamides, benzodiazepines and other aromatic or non-aromatic heterocycles(see Estiarte et al., Burgers Medicinal Chemistry, 6^(th) edition,Volume 1, Part 4, John Wiley & Sons, New York (2002)). Modified aminoacids are often connected to the peptide with at least one of the abovementioned functional groups instead of an amide bond. Non-proteinogenicamino acids include, but are not limited, to β-alanine (Bal), norvaline(Nva), norleucine (Nle), 4-aminobutyric acid (γ-Abu), 2-aminoisobutyricacid (Aib), 6-aminohexanoic acid (ε-Ahx), ornithine (Orn),hydroxyproline (Hyp), taurine, sarcosine, citrulline (Cit), cysteic acid(Coh), cyclohexylalanine (Cha), methioninesulfoxide (Meo),methioninesulfone (Moo), homoserinemethylester (Hsm), propargylglycine(Eag), 5-fluorotryptophan (5Fw), 6-fluorotryptophan (6Fw),3′,4′-dimethoxyphenyl-alanine (Ear), 3′,4′-difluorophenylalanine (Dff),4′-fluorophenyl-alanine (Pff), 1-naphthyl-alanine (1Ni),1-methyltryptophan (1Mw), penicillamine (Pen), homoserine (Hse),t-butylglycine, t-butylalanine, phenylglycine (Phg), benzothienylalanine(Bta), L-homo-cysteine (Hcy), N-methyl-phenylalanine (Nmf),2-thienylalanine (Thi), 3,3-diphenylalanine (Ebw), homophenylalanine(Hfe) and S-benzyl-L-cysteine (Ece). The structures of many of thenon-proteinogenic amino acids are provided in Table 2. These and othernon-proteinogenic amino acids may exist as D- or L-isomers. Examples ofmodified linkers include, but are not limited to, the flexible linker4,7,10-trioxa-1,13-tridecanediamine (Ttds), glycine, 6-aminohexanoicacid, beta-alanine (Bal), pentynoic acid (Pyn), and combinations ofTtds, glycine, 6-aminohexanoic acid and Bal.

Homologs of the amino acids constituting the peptides of the inventionmay be as set forth in Table 3. In any embodiment, one or more aminoacids of the TFPI-binding peptide are substituted with a homolog.

TABLE 3 Amino Acid Exemplary homologs A Aib, Bal, Eag, Nma, Abu, G, M,Nva, Nle C S, A, Hcy, M, L, I, V, Nmc, β-Cysteine D E, Homoglutamicacid, γ-Hydroxy-glutamic acid, γ- Carboxy-glutamic acid, Nmd, β-Asparticacid, N, Q, Cysteic acid E D, Glu, Homoglutamic acid, γ-Hydroxy-glutamicacid, γ- Carboxy-glutamic acid, α-Aminoadipic acid, Nme, β- glutamicacid, Q, N, Cysteic acid F Hfe, Nmf, β-Phenylalanine, Phg, Bhf,Thienylalanine, Benzothienylalanine, Bromophenylalanine,Iodophenylalanione, Chlorophenylalanine, Methylphenylalanine,Nitrophenylalanine, Y, W, Naphtylalanine, Tic G A, Nmg H Nmh,1-Methylhistidine, 3-Methylhistidine, Thienylalanine I L, V, Hle, Nva,Nle, β-Isoleucine, Nml, M, Nmi K Nmk, R, Nmr, β-Lysine, Dab, Dap,β-(1-Piperazinyl)- alanine, 2,6-Diamino-4-hexynoic acid,delta-Hydroxy-lysine, Har, omega-Hydroxy-norarginine,omega-Amino-arginine, omega-Methyl-arginine, β-(2-Pyridyl)-alanine,β-(3- Pyridyl)-alanine, 3-Amino-tyrosine, 4-Amino-phenylalanine, Hci,Cit L I, V, Hle, Nle, Nva, β-Isoleucine, Nml, M M I, V, Hle, Nva, R,Har, Nmm, Methioninesulfone N Nmn, β-Asparagine, Q, Nmq, β-Glutamine,Cys(3-propionic acid amide)-OH, Cys(O2-3-propionic acid amide)-OH PAzetidine-2-carboxylic acid, Hyp, α-Methyl-methionine, 4-Hydroxy-piperidine-2-carboxylic acid, Pip, α-Methyl-Pro Q N, Nmn, Nmq,β-Glutamine, Cys(3-propionic acid amide)-OH, Cys(O2-3-propionic acidamide)-OH R Nmk, K, Nmr, β-Lysine, Dab, Dap, Orn, β-(1-Piperazinyl)-alanine, 2,6-Diamino-4-hexynoic acid, delta-Hydroxy-lysine, Har,omega-Hydroxy-norarginine, omega-Amino-arginine, omega-Methyl-arginine,β-(2-Pyridyl)-alanine, β-(3- Pyridyl)-alanine, 3-Amino-tyrosine,4-Amino-phenylalanine, Hci, Cit, Hle, L, Nle, M S T, Hse, β-Serine, C,β-Cyano-alanine, allo-Threonine T S, Homothreonine, β-Threonine,allo-Threonine V L, I, Hle, Nva, Nle, β-Valine, Nmv, M, Nmi, Nml W Nmw,β-Tryptophan, F, Hfe, Nmf, β-Phenylalanine, Phg, Bhf, Thienylalanine,Benzothienylalanine, Bromophenylalanine, Iodophenylalanine,Chlorophenylalanine, Methylphenylalanine, Nitrophenylalanine, Y,Naphtylalanine, Tic Y Nmy, β-Tyrosine,, F, Hfe, Nmf, β-Phenylalanine,Phg, Bhf, Thienylalanine, Benzothienylalanine, Bromophenylalanine,Iodophenylalanine, Chlorophenylalanine, Methylphenylalanine,Nitrophenylalanine, W, Naphtylalanine, Tic

Derivatives also include peptides comprising amino acids having modifiedsubstituents, such as amino acids modified by halogenation with, e.g.,fluorine, chlorine, iodine, or bromine. In some embodiments, theTFPI-binding peptide comprises a halogenated aromatic amino acid, suchas phenylalanine.

In some embodiments, the peptide (CO—NH) linkages joining amino acidswithin the peptide of the invention are reversed to create a“retro-modified” peptide, i.e., a peptide comprising amino acid residuesassembled in the opposite direction (NH—CO bonds) compared to thereference peptide. The retro-modified peptide comprises the same aminoacid chirality as the reference peptide. An “inverso-modified” peptideis a peptide of the invention comprising amino acid residues assembledin the same direction as a reference peptide, but the chirality of theamino acids is inverted. Thus, where the reference peptide comprisesL-amino acids, the “inverso-modified” peptide comprises D-amino acids,and vice versa. Inverso-modified peptides comprise CO—NH peptide bonds.A “retro-inverso modified” peptide refers to a peptide comprising aminoacid residues assembled in the opposite direction and which haveinverted chirality. A retro-inverso analogue has reversed termini andreversed direction of peptide bonds (i.e., NH—CO), while approximatelymaintaining the side chain topology found in the reference peptide.Retro-inverso peptidomimetics are made using standard methods, includingthe methods described in Meziere et al, J. Immunol., 159, 3230-3237(1997), incorporated herein by reference. Partial retro-inverso peptidesare peptides in which only part of the amino acid sequence is reversedand replaced with enantiomeric amino acid residues.

TFPI-binding peptides of the invention (including TFPI inhibitorpeptides) are made in a variety of ways. In one aspect, the peptides aresynthesized by solid phase synthesis techniques including thosedescribed in Merrifield, J. Am. Chem. Soc., 85, 2149 (1963); Davis etal., Biochem. Intl., 10, 394-414 (1985); Larsen et al., J. Am. Chem.Soc., 115, 6247 (1993); Smith et al., J. Peptide Protein Res., 44, 183(1994); O'Donnell et al., J. Am. Chem. Soc., 118, 6070 (1996); Stewartand Young, Solid Phase Peptide Synthesis, Freeman (1969); Finn et al.,The Proteins, 3^(rd) ed., vol. 2, pp. 105-253 (1976); and Erickson etal., The Proteins, 3^(rd) ed., vol. 2, pp. 257-527 (1976).Alternatively, the TFPI-binding peptide (e.g., the TFPI-inhibitorypeptide) is expressed recombinantly by introducing a nucleic acidencoding a TFPI-binding peptide (e.g., a TFPI-inhibitory peptide) intohost cells, which are cultured to express the peptide. Such peptides arepurified from the cell culture using standard protein purificationtechniques.

The invention also encompasses a nucleic acid comprising a nucleic acidsequence encoding a peptide of the invention. Methods of preparing DNAand/or RNA molecules are well known in the art. In one aspect, a DNA/RNAmolecule encoding a peptide provided herein is generated using chemicalsynthesis techniques and/or using polymerase chain reaction (PCR). Ifdesired, a peptide coding sequence is incorporated into an expressionvector. One of ordinary skill in the art will appreciate that any of anumber of expression vectors known in the art are suitable in thecontext of the invention, such as, but not limited to, plasmids,plasmid-liposome complexes, and viral vectors. Any of these expressionvectors are prepared using standard recombinant DNA techniques describedin, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 2dedition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, New York, N.Y. (1994).Optionally, the nucleic acid is operably linked to one or moreregulatory sequences, such as a promoter, activator, enhancer, capsignal, polyadenylation signal, or other signal involved with thecontrol of transcription or translation.

Any of the peptides (or peptide complexes) of the invention or nucleicacids encoding the peptides also is provided in a composition (e.g., apharmaceutical composition). In this regard, the peptide (or peptidecomplex) is formulated with a physiologically-acceptable (i.e.,pharmacologically-acceptable) carrier, buffer, excipient, or diluent, asdescribed further herein. Optionally, the peptide is in the form of aphysiologically acceptable salt, which is encompassed by the invention.“Physiologically acceptable salts” means any salts that arepharmaceutically acceptable. Some examples of appropriate salts includeacetate, hydrochloride, hydrobromide, sulfate, citrate, tartrate,glycolate, and oxalate. If desired, the composition comprises one ormore additional pharmaceutically-effective agents.

The peptide provided herein optionally inhibits at least one TFPI-1(e.g., TFPI-1α or TFPI-1β) activity such as, but not limited to, anactivity that downregulates the blood coagulation cascade. Without beingbound by any specific mechanism of action, a proposed mechanism ofinhibition may involve preventing formation of the quaternaryTF-FVIIA-FXA-TFPI complex. The peptide may inhibit binding(competitively or allosterically) of TFPI to FXa (e.g., inhibit bindingof TFPI Kunitz domain 2 to Factor Xa or interrupt binding of TFPI Kunitzdomain 1 to an exosite of Factor Xa), the TF/FVIIa complex (e.g.,inhibit binding of TFPI Kunitz domain 1 to the TF/FVIIa complex), TFalone, and/or FVIIa alone. With TFPI activity diminished, TF and FVIIaare free to activate FX which, in turn, enhances conversion ofprothrombin to thrombin. Surprisingly, in one embodiment, the peptide ofthe invention that binds Kunitz domain 1 interferes with TFPI-mediatedinhibition of FXa. Thus, the invention provides a method of, e.g.,inhibiting TFPI-mediated downregulation of the extrinsic and/or commonpathway of the coagulation cascade and/or enhancing FXa-mediatedconversion of prothrombin to thrombin, by administering to a subject apeptide described herein that binds Kunitz domain 1.

In one aspect, the peptide of the invention exhibits TFPI antagonisticactivity in model and/or plasmatic systems. An exemplary model systemfor determining TFPI-inhibitory activity is the extrinsic tenase assay,which tests the ability of candidate peptides to restore extrinsiccomplex-mediated FX activation in the presence of TFPI (which is anatural inhibitor of the FX activation reaction) (see, e.g., Lindhout etal., Thromb. Haemost., 74, 910-915 (1995)). Another model system forcharacterizing TFPI-inhibitory activity is the FXa inhibition assay,wherein FXa activity is measured in the presence of TFPI (see Sprecheret al., PNAS, 91, 3353-3357 (1994)). The extrinsic tenase assay and theFXa inhibition assay are further described in Example 3. Optionally, thepeptide of the invention enhances FX activation in the presence of TFPIwith a half maximal effective concentration (EC₅₀) of less than or equalto 1×10⁻⁴M, less than or equal to 1×10⁻⁵M, less than or equal to1×10⁻⁶M, or less than or equal to 1×10⁻⁷M.

In one aspect, TFPI-antagonist activity is characterized in aplasma-based assay. Thrombin formation is triggered in plasmasubstantially lacking FVIII or FIX activity (e.g., the residualcoagulation factor activity is lower than 1%) in the presence of acandidate peptide. Thrombin formation can be detected using afluorogenic or chromogenic substrate, as described in Example 4. Asystem for measuring thrombin activity is provided by Thrombinoscope BV(Maastricht, The Netherlands). Prothrombin conversion is measured using,e.g., a Thrombograph™ (Thermo Scientific, Waltham, Mass.), and theresulting data is compiled into a Calibrated Automatic Thrombogramgenerated by Thrombinoscope™ software available from Thrombinoscope BV.In certain embodiments, the TFPI-inhibitory peptide increases the amountof peak thrombin generated during the assay and/or decreases the timerequired to achieve peak thrombin formation. For example, the peptideimproves TFPI-regulated thrombin generation in the absence of FVIII(e.g., in FVIII-depleted plasma) to at least 1% of the level ofTFPI-dependent thrombin generation in normal plasma. Generally, normal(unafflicted) plasma contains about 0.5 U/mL to about 2 U/mL FactorVIII. Accordingly, in some instances, a TFPI-binding peptide (e.g.,TFPI-inhibitory peptide) will enhance thrombin formation in the absenceof FVIII to at least about 1% of that observed in the presence of 0.5U/mL to 2 U/mL FVIII. In further embodiments, the peptide enhancesthrombin formation in the absence of Factor VIII to at least about 2%,at least about 3%, at least about 5%, at least about 7%, or at leastabout 10% of the level of thrombin formation in normal plasma, i.e., inthe presence of physiological levels of Factor VIII. In various aspects,the peptide is administered to an animal model of thrombin deficiency orhemophilia to characterize TFPI inhibitory activity in vivo. Such invivo models are known in the art and include for example, miceadministered anti-FVIII antibodies to induce hemophilia A (Tranholm etal., Blood, 102, 3615-3620 (2003)); coagulation factor knock-out modelssuch as, but not limited to, FVIII knock-out mice (Bi et al., Nat.Genet., 10(1), 119-121 (1995)) and FIX knock-out mice (Wang et al.,PNAS, 94(21), 11563-66 (1997)); induced hemophilia-A in rabbits (Shen etal., Blood, 42(4), 509-521 (1973)); and Chapel Hill HA dogs (Lozier etal., PNAS, 99, 12991-12996 (2002)).

Various peptides bind TFPI from any source including, but not limitedto, mouse, rat, rabbit, dog, cat, cow, horse, pig, guinea pig, andprimate. In one embodiment, the peptide binds human TFPI. Optionally,the TFPI-binding peptide (e.g., TFPI-inhibitory peptide) binds TFPI frommore than one species (i.e., the peptide is cross-reactive amongmultiple species). In certain aspects, the peptide binds TFPI with adissociation constant (K_(D)) of less than or equal to 1×10⁻⁴M, lessthan or equal to 1×10⁻⁵M, less than or equal to 1×10⁻⁶M, or less than orequal to 1×10⁻⁷M. Affinity may be determined using, for example andwithout limitation, any one, two, or more of a variety of techniques,such as affinity ELISA assay, a competitive ELISA assay, and/or surfaceplasmon resonance (BIAcore™) assay. When characterized using acompetitive (IC₅₀) ELISA assay, the peptide of the invention optionallydemonstrates an IC₅₀ of less than or equal to about 50,000 nM. Forexample, the peptide demonstrates an IC₅₀ of less than or equal to about10,000 nM, such as an IC₅₀ of less than or equal to about 5,000 nM, lessthan or equal to about 1,000 nM, or less than or equal to about 500 nM.In one aspect, the peptide demonstrates an IC₅₀ of less than or equal toabout 250 nM, less than or equal to about 100 nM, less than or equal toabout 50 nM, or less than or equal to about 10 nM. Exemplary peptidesand their IC₅₀ values are provided in FIGS. 32-39; in some instances,the peptides are classified into Groups A, B, C, D, E, F, and G (seeTable 4 in Example 1) based on their IC₅₀ values. In various aspects,the invention provides peptides falling within Groups A, B, C, D, E, F,and/or G as defined in Table 4. Affinity may also be determined by akinetic method or an equilibrium/solution method. Such methods aredescribed in further detail herein or known in the art.

Another suitable assay for characterizing the inventive peptides is ak_(off) assay, which examines a peptide's release from TFPI. The k_(off)assay result is not the dissociation rate constant, but a percentage ofcompetitor peptide blocked from TFPI binding by a test peptide after anincubation period with TFPI. An exemplary k_(off) assay includes thefollowing steps: 1) incubation of a TFPI-coated microtiter plate with anamount of test peptide resulting in approximately 90% TFPI occupation;2) removal of unbound test peptide; 3) addition of a biotinylated tracer(i.e., competitor) peptide that competes with the test peptide forbinding to TFPI; 4) incubation for a period of time during which bindingsites released by the test peptide is occupied by the tracer; 5) removalof unbound tracer and test peptide; and 6) detection of bound tracer bya chromogenic reaction using streptavidin-horseradish peroxidaseconjugate. The resulting signal is indicative of binding sites freed bythe test peptide. A test peptide that does not dissociate from TFPIduring the incubation period yields a weaker signal compared to ananalyte that dissociates completely.

As with all binding agents and binding assays, one of skill in the artrecognizes that the various moieties to which a binding agent should notdetectably bind in order to be biologically (e.g., therapeutically)effective would be exhaustive and impractical to list. Therefore, theterm “specifically binds” refers to the ability of a peptide to bindTFPI with greater affinity than it binds to an unrelated control proteinthat is not TFPI. For example, the peptide may bind to TFPI with anaffinity that is at least, 5, 10, 15, 25, 50, 100, 250, 500, 1000, or10,000 times greater than the affinity for a control protein. In someembodiments, the peptide binds TFPI with greater affinity than it bindsto an “anti-target,” a protein or other naturally occurring substance inhumans to which binding of the peptide might lead to adverse effects.Several classes of peptides or proteins are potential anti-targets.Because TFPI-inhibitory peptides exert their activity in the bloodstream and/or at the endothelium, plasma proteins represent potentialanti-targets. Proteins containing Kunitz domains (KDs) are potentialanti-targets because KDs of different proteins share a significantsimilarity. Tissue Factor Pathway Inhibitor-2 (TFPI-2) is highly similarto TFPI-1α and, like TFPI-1α, contains KDs (Sprecher et al., PNAS, 91,3353-3357 (1994)). Thus, in one aspect, the peptide of the inventionbinds to TFPI with an affinity that is at least 5, 10, 15, 25, or 50times greater than the affinity for an anti-target, such as TFPI-2.

Optionally, the TFPI-binding peptide demonstrates one or more desiredcharacteristics described herein, and the amino acid sequence of apeptide can be modified to optimize binding, stability, and/or activity,if desired. An exemplary TFPI-binding peptide binds TFPI with a K_(D) ofless than or equal to 20 nM and/or exhibits a binding affinity for TFPIthat is at least 100 times greater than the binding affinity for ananti-target. Alternatively or in addition, the TFPI-binding peptideenhances FX activation in the presence of TFPI with an EC₅₀ (as measuredusing any suitable assay, such as the assays described here) of lessthan or equal to 50 nM and/or enhances thrombin formation in the absenceof Factor VIII to at least about 20% (e.g., 40%) of the level ofthrombin formation in plasma containing physiological levels of FactorVIII. Alternatively or in addition, the TFPI-binding peptide achieves adesired level of plasma stability (e.g., 50% or more of a dose remainsin plasma after 12 hours) and/or demonstrates a desired half life invivo (e.g., at least two, three, four, five, six, seven, eight, nine, orten hours). Alternatively or in addition, the TFPI-binding peptideexhibits a desired level of bioavailability, such as a desired level ofbioavailability following subcutaneous administration (e.g., greaterthan or equal to 5%, 10%, 15%, 20%, 25%, 30%, or 50%) and/ordemonstrates a desired level of TFPI-inhibitory activity at a given dosein vivo.

The invention further includes a method of inhibiting TFPI-1. The methodcomprises contacting TFPI with a TFPI-binding peptide as describedherein. Any degree of TFPI-activity inhibition is contemplated. Forexample, a TFPI-binding peptide (e.g., TFPI-inhibitory peptide) reducesTFPI-inhibition of the extrinsic pathway at least about 5% (e.g., atleast about 10%, at least about 25%, or at least about 30%). In someembodiments, the TFPI-binding peptide (e.g., TFPI-inhibitory peptide)reduces TFPI activity within the extrinsic pathway at least about 50%,at least about 75%, or at least about 90% compared to TFPI activity inthe absence of the peptide.

In one aspect of the invention, TFPI-binding peptides are used to detectand/or quantify TFPI in vivo or in vitro. An exemplary method ofdetecting and/or quantifying TFPI in a sample comprises (a) contacting asample with a TFPI-binding peptide of the invention, and (b) detectingbinding of the TFPI-binding peptide to TFPI.

The invention further includes a method for targeting biologicalstructures (including, but not limited to, cell surfaces and endotheliallining) where TFPI is located. The method comprises contacting thebiological structure (e.g., including, without limitation, a celldisplaying TFPI on the cell surface) with a TFPI-binding peptidedescribed herein, optionally conjugated to a moiety that adds additionalfunctionality to the peptide. The moiety can be a dye (such as afluorescence dye), a radionuclide or a radionuclide-containing complex,a protein (e.g., an enzyme, a toxin, or an antibody) or a cytotoxicagent. For example, the peptide is linked or conjugated to an effectormoiety that facilitates peptide detection and/or purification and/orcomprises therapeutic properties. In one aspect, the TFPI-bindingpeptide or peptide conjugate is administered to a mammal to target aTFPI-displaying cell within the mammal. Optionally, the method furthercomprises detecting binding of the TFPI-binding peptide to TFPI. Themethod is useful for therapy and diagnosis of disease where TFPI is asuitable diagnostic marker or TFPI-expressing cells are a target for atherapeutic approach.

Peptide-TFPI complexes are directly or indirectly detected. Detectionmoieties are widely used in the art to identify biological substancesand include, for example, dye (e.g., fluorescent dye), radionuclides andradionuclide-containing complexes, and enzymes. In some aspects,peptide-TFPI binding is detected indirectly. In this regard, the peptideis optionally contacted with an interaction partner that binds thepeptide of invention without significantly interfering with peptide-TFPIbinding, and the interaction partner is detected. Exemplary interactionpartners include, but are not limited to, antibodies, antigen-bindingantibody fragments, anticalins and antibody mimetics, aptamers,streptavidin, avidin, neutravidin, and spiegelmers. Optionally, theinteraction partner comprises a detection moiety to facilitate detectionof an interaction partner-peptide complex. The TFPI-binding peptide is,in some embodiments, modified to facilitate binding of an interactionpartner. For example, in one aspect, the TFPI-binding peptide isconjugated to biotin, which is bound by an interaction partnercomprising streptavidin. An exemplary interaction partner comprisesstrepavidin fused to horseradish peroxidase, which is detected in, e.g.,an ELISA-like assay. Alternatively, the TFPI-binding peptide is modifiedto include an antibody epitope, and binding of the correspondingantibody to the peptide-TFPI complex is detected. Methods of detecting,e.g., antibodies and fragments thereof, are well understood in the art.

Peptide-TFPI complexes and interaction partner-peptide complexes areidentified using any of a number of methods, such as, but not limitedto, biochemical assays (e.g., enzymatic assays), spectroscopy (e.g.,detection based on optical density, fluorescence, FRET, BRET, TR-FRET,fluorescence polarization, electrochemoluminescence, or NMR), positronemission tomography (PET), and single Photon Emission ComputedTomography (SPECT). Detectable moieties that facilitate fluorescencedetection of peptide-TFPI complexes or interaction partner-peptidecomplexes include, but are not limited to, fluorescein, Alexa Fluor®350, Marina Blue™, Cascade Yellow™, Alexa Fluor® 405, Pacific Blue™,Pacific Orange™, Alexa Fluor® 430, Alexa Fluor® 488, Oregon Green® 488,Alexa Fluor® 500, Oregon Green® 514, Alexa Fluor® 514, Alexa Fluor® 532,Alexa Fluor® 555, Tetramethylrhodamine, Alexa Fluor® 546, Rhodamine B,Rhodamine Red™-X, Alexa Fluor® 568, Alexa Fluor® 594, Texas Red®, TexasRed®-X, Alexa Fluor® 610, Alexa Fluor® 633, Alexa Fluor® 635, AlexaFluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, AlexaFluor® 750, B-Phycoerythrin, R-Phycoerythrin, Allophycocyanin, BODIPY®,Cy3, Cy5, TAMRA, and fluorescent proteins (GFP and derivatives thereof).An example of a TFPI-binding peptide comprising a fluorescent detectionmoiety is JBT2454 (FAM-Ttds-FQSKpNVHVDGYFERL-Aib-AKL-NH2 (SEQ ID NO:4171)), which is labeled with 5,6-carboxyfluoresceine.

Radioactive labels also are used to detect biological materials (e.g.,TFPI, TFPI-binding peptides, or TFPI-binding peptide-TFPI complexes),and, in some instances, are attached to peptides or interaction partnersusing a chelator, such as (but not limited to) EDTA (ethylene diaminetetra-acetic acid), DTPA (diethylene triamine pentaacetic acid), CDTA(cyclohexyl 1,2-diamine tetra-acetic acid), EGTA(ethyleneglycol-O,O′-bis(2-aminoethyl)-N,N,N′,N′-tetra-acetic), HBED(N,N-bis(hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid), TTHA(triethylene tetramine hexa-acetic acid), DOTA(1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetra-acetic acid), HEDTA(hydroxyethyldiamine triacetic acid), or TETA(1,4,8,11-tetra-azacyclotetradecane-N,N′,N″,N″″-tetra-acetic acid).Examples of radioactive labels include ^(99m)Tc, ²⁰³Pb, ⁶⁶Ga, ⁶⁷Ga,⁶⁸Ga, ⁷²A5, ¹¹¹In, ^(113m)In, ^(114m)In, ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁵²Fe,^(52m)Mn, ⁵¹Cr, ¹⁸⁶Re. ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ^(117m)Sn, ¹²¹Sn,¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁴⁹Tb, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm,¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, and¹¹¹Ag. Paramagnetic metals also are detectable moieties that aresuitable for attachment to TFPI-binding peptides or interactionpartners, optionally via chelator complex. Examples of paramagneticmetals include, for example, Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sm, Yb, Gd,Tb, Dy, Ho, and Er.

TFPI-binding peptides, themselves, are, in some aspects, modified toinclude one or more amino acids with detectable substituents ornuclides. In this regard, in one embodiment, the TFPI-binding peptidecomprises at least one amino acid comprising a detectable isotope (e.g.,¹³C, ¹⁴C, ³⁵S, ³H, ¹⁸O or ¹⁵N), and/or an amino acid that is halogenatedwith, e.g., ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br or ⁸²Br. Aminoacids suitable for halogenation include, but are not limited to,tyrosine and tryptophan.

The invention also provides a method for diagnosing a subject sufferingfrom a disease or disorder, or at risk of suffering from a disease ordisorder, wherein the disease or disorder is associated with or causedby aberrant TFPI activity. The method comprises administering to thesubject the TFPI-binding peptide and detecting the TFPI-peptide complex.In some instances, the peptide is conjugated to a detectable moiety, andthe method comprises detecting the detectable moiety. Exemplarydetectable moieties are described herein. In other instances, the methodcomprises administering to the subject a TFPI-binding peptideinteraction partner that binds the TFPI-binding peptide, and detectingthe interaction partner. If desired, the interaction partner comprisesor is conjugated to a detectable moiety, and the detectable moiety isdetected. The presence of the detectable moiety indicates the presenceof TFPI, thereby allowing diagnosis of a disease or disorder associatedwith TFPI (e.g., a disease or disorder which (i) can be treated byinhibiting TFPI or (ii) comprises symptoms which can be ameliorated orprevented by inhibiting TFPI). If administration of the peptide to thesubject is not desired, a biological sample is obtained from thesubject, contacted with the TFPI-binding peptide as described herein,and TFPI-peptide complexes are detected.

The peptides of the invention bind TFPI and, therefore, are useful forpurifying TFPI or recombinant TFPI from a biological sample (e.g., abiological fluid, such as serum), fermentation extract, tissuepreparations, culture medium, and the like. The invention includesmethods of using the TFPI-binding peptide in the commercial productionof TFPI or in a method of characterizing TFPI molecules. For example,the invention includes a method of purifying TFPI. The method comprisescontacting a sample containing TFPI with a peptide as defined hereinunder conditions appropriate to form a complex between TFPI and thepeptide; removing the complex from the sample; and, optionally,dissociating the complex to release TFPI. Exemplary conditionsappropriate to form a complex between TFPI and the peptide are disclosedin the Examples, and such conditions can be easily modified todissociate the TFPI-peptide complex. In some embodiments, the peptide isimmobilized to a support, e.g., a solid support, to facilitate recoveryof TFPI. For example, in one embodiment, the peptide is immobilized tochromatography stationary phase (e.g., silica, affinity chromatographybeads, or chromatography resins), a sample comprising TFPI is applied tothe stationary phase such that TFPI-peptide complexes are formed, theremainder of the sample is removed from the stationary phase, and TFPIis eluted from the stationary phase. In this regard, the peptides of theinvention are, in one aspect, suitable for use in affinitychromatography techniques.

A method of enhancing thrombin formation in a clotting factor-deficientsubject also is provided. The method comprises administering to thesubject a peptide provided herein under conditions effective to inhibitTFPI. In this regard, the TFPI-binding peptide is administered in anamount and under conditions effective to enhance thrombin formation inthe subject. By “clotting factor-deficient” is meant that the subjectsuffers from a deficiency in one or more blood factors required forthrombin formation, such as FVIII, FIX, or FXI. Indeed, in oneembodiment, the subject is deficient in FVIII. Alternatively or inaddition, the subject is deficient in Factor IX. Clotting factordeficiencies are identified by examining the amount of factor in aclinical sample. Practitioners classify hemophilia according to themagnitude of clotting factor deficiency. Subjects suffering from mildhemophilia have approximately 5% to 30% of the normal amount (1 U/ml) ofFactor VIII or Factor IX. Moderate hemophilia is characterized byapproximately 1% to 5% of normal Factor VIII, Factor IX, or Factor XIlevels, while subjects suffering from severe hemophilia have less than1% of the normal amount of Factor VIII, Factor IX, or Factor XI.Deficiencies can be identified indirectly by activated partialthromboplastin time (APTT) testing. APTT testing measures the length oftime required for a blood clot to form, which is longer for patientswith Factor VIII Deficiency (hemophilia A), Factor IX Deficiency(hemophilia B), and Factor XI Deficiency (hemophilia C) compared topatients with normal clotting factor levels. Almost 100% of patientswith severe and moderate Factor VIII deficiency can be diagnosed with anAPTT. The invention further includes enhancing thrombin formation in asubject that does not suffer from a clotting factor deficiency. Themethod comprises administering to a subject (e.g., a subject comprisingnormal, physiological levels of clotting factor) a peptide providedherein under conditions effective to enhance thrombin formation.

In one aspect, the TFPI-binding peptide is used for increasing bloodclot formation in a subject. The method of increasing blood clotformation comprises administering to the subject a peptide describedherein in an amount and under conditions effective to increase bloodclot formation. It will be appreciated that the method need notcompletely restore the coagulation cascade to achieve a beneficial(e.g., therapeutic) effect. Any enhancement or increase in thrombin orblood clot formation that reduces the onset or severity of symptomsassociated with clotting factor deficiencies is contemplated. Methods ofdetermining the efficacy of the method in promoting thrombin formationand blood clotting are known in the art and described herein.

The invention further includes a method of treating a blood coagulationdisorder in a subject, the method comprising administering to thesubject one or more TFPI-binding peptides (or peptide complex(es)), suchas any one or more of the peptides described herein, in an amount andunder conditions effective to treat the blood coagulation disorder inthe subject. In one aspect, the peptide is a recombinant or syntheticpeptide that inhibits TFPI activity. “Coagulation disorders” includebleeding disorders caused by deficient blood coagulation factor activityand deficient platelet activity. Blood coagulation factors include, butare not limited to, Factor V (FV), FVII, FVIII, FIX, FX, FXI, FXIII, FII(responsible for hypoprothrombinemia), and von Willebrand's factor.Factor deficiencies are caused by, for instance, a shortened invivo-half life of the factor, altered binding properties of the factor,genetic defects of the factor, and a reduced plasma concentration of thefactor. Coagulation disorders can be congenital or acquired. Potentialgenetic defects include deletions, additions and/or substitution withina nucleotide sequence encoding a clotting factor whose absence,presence, and/or substitution, respectively, has a negative impact onthe clotting factor's activity. Coagulation disorders also stem fromdevelopment of inhibitors or autoimmunity (e.g., antibodies) againstclotting factors. In one example, the coagulation disorder is hemophiliaA. Alternatively, the coagulation disorder is hemophilia B or hemophiliaC.

Platelet disorders are caused by deficient platelet function orabnormally low platelet number in circulation. Low platelet count may bedue to, for instance, underproduction, platelet sequestration, oruncontrolled patent destruction. Thrombocytopenia (plateletdeficiencies) may be present for various reasons, including chemotherapyand other drug therapy, radiation therapy, surgery, accidental bloodloss, and other disease conditions. Exemplary disease conditions thatinvolve thrombocytopenia are: aplastic anemia; idiopathic or immunethrombocytopenia (ITP), including idiopathic thrombocytopenic purpuraassociated with breast cancer; HIV-associated ITP and HIV-relatedthrombotic thrombocytopenic purpura; metastatic tumors which result inthrombocytopenia; systemic lupus erythematosus, including neonatal lupussyndrome splenomegaly; Fanconi's syndrome; vitamin B12 deficiency; folicacid deficiency; May-Hegglin anomaly; Wiskott-Aldrich syndrome; chronicliver disease; myelodysplastic syndrome associated withthrombocytopenia; paroxysmal nocturnal hemoglobinuria; acute profoundthrombocytopenia following C7E3 Fab (Abciximab) therapy; alloimmunethrombocytopenia, including maternal alloimmune thrombocytopenia;thrombocytopenia associated with antiphospholipid antibodies andthrombosis; autoimmune thrombocytopenia; drug-induced immunethrombocytopenia, including carboplatin-induced thrombocytopenia andheparin-induced thrombocytopenia; fetal thrombocytopenia; gestationalthrombocytopenia; Hughes' syndrome; lupoid thrombocytopenia; accidentaland/or massive blood loss; myeloproliferative disorders;thrombocytopenia in patients with malignancies; thromboticthrombocytopenia purpura, including thrombotic microangiopathymanifesting as thrombotic thrombocytopenic purpura/hemolytic uremicsyndrome in cancer patients; post-transfusion purpura (PTP); autoimmunehemolytic anemia; occult jejunal diverticulum perforation; pure red cellaplasia; autoimmune thrombocytopenia; nephropathia epidemica;rifampicin-associated acute renal failure; Paris-Trousseauthrombocytopenia; neonatal alloimmune thrombocytopenia; paroxysmalnocturnal hemoglobinuria; hematologic changes in stomach cancer;hemolytic uremic syndromes (e.g., uremic conditions in childhood); andhematologic manifestations related to viral infection includinghepatitis A virus and CMV-associated thrombocytopenia. Plateletdisorders also include, but are not limited to, Von Willebrand Disease,paraneoplastic platelet dysfunction, Glanzman's thrombasthenia, andBernard-Soulier disease. Additional bleeding disorders amenable totreatment with a TFPI-binding peptide (e.g., TFPI-inhibitory peptide)include, but are not limited to, hemorrhagic conditions induced bytrauma; a deficiency in one or more contact factors, such as FXI, FXII,prekallikrein, and high molecular weight kininogen (HMWK); vitamin Kdeficiency; a fibrinogen disorder, including afibrinogenemia,hypofibrinogenemia, and dysfibrinogenemia; and alpha2-antiplasmindeficiency. In one embodiment, the TFPI-binding peptide (e.g.,TFPI-inhibitory peptide) is used to treat excessive bleeding, such asexcessive bleeding caused by surgery, trauma, intracerebral hemorrhage,liver disease, renal disease, thrombocytopenia, platelet dysfunction,hematomas, internal hemorrhage, hemarthroses, hypothermia, menstruation,pregnancy, and Dengue hemorrhagic fever. All of the above are considered“blood coagulation disorders” in the context of the disclosure.

In one aspect, the TFPI-binding peptide (e.g., TFPI-inhibitory peptide)of the invention is used to reverse the effects (in whole or in part) ofone or more anticoagulants in a subject. Numerous anticoagulants areknown in the art and include, for instance, heparin; coumarinderivatives, such as warfarin or dicumarol; TFPI; AT III; lupusanticoagulant; nematode anticoagulant peptide (NAPc2); FVIIa inhibitors;active-site blocked FVIIa (FVIIai); active-site blocked FIXa (FIXai);FIXa inhibitors; FXa inhibitors, including fondaparinux, idraparinux,DX-9065a, and razaxaban (DPC906); active-site blocked FXa (FXai);inhibitors of FVa or FVIIIa, including activated protein C (APC) andsoluble thrombomodulin; thrombin inhibitors, including hirudin,bivalirudin, argatroban, and ximelagatran; and antibodies or antibodyfragments that bind a clotting factor (e.g., FV, FVII, FVIII, FIX, FX,FXIII, FII, FXI, FXII, von Willebrand factor, prekallikrein, or highmolecular weight kininogen (HMWK)).

As used herein, “treating” and “treatment” refers to any reduction inthe severity and/or onset of symptoms associated with a bloodcoagulation disorder. Accordingly, “treating” and “treatment” includestherapeutic and prophylactic measures. One of ordinary skill in the artwill appreciate that any degree of protection from, or amelioration of,a blood coagulation disorder or symptom associated therewith isbeneficial to a subject, such as a human patient. The quality of life ofa patient is improved by reducing to any degree the severity of symptomsin a subject and/or delaying the appearance of symptoms. Accordingly,the method in one aspect is performed as soon as possible after it hasbeen determined that a subject is at risk for developing a bloodcoagulation disorder (e.g., a deficiency in a clotting factor (e.g.,FVIII, FIX, or FXI) is detected) or as soon as possible after a bloodcoagulation disorder (e.g., hemophilia A, hemophilia B, or hemophilia C)is detected. In an additional aspect, the peptide is administered toprotect, in whole or in part, against excessive blood loss during injuryor surgery.

In view of the above, the invention provides a peptide (or peptidecomplex) for use in a method for the treatment of a subject, such as amethod for the treatment of a disease where the inhibition of TFPI isbeneficial. In one aspect, the disease or disorder is a bloodcoagulation disorder. The subject is suffering from a disease ordisorder or is at risk from suffering from a disease or disorder (oradverse biological event, such as excessive blood loss). The methodcomprises administering to the subject the peptide (or peptide complex)of the invention in an amount and under conditions effective to treat orprevent, in whole or in part, the disease or disorder. The inventionfurther provides a peptide (or peptide complex) for use in themanufacture of a medicament. For example, the peptide (or peptidecomplex) can be used in the manufacture of a medicament for thetreatment of a blood coagulation disorder, as described in detailherein.

In some embodiments, it is advantageous to administer to a subject anucleic acid comprising a nucleic acid sequence encoding a peptidecomplex or peptide (e.g., TFPI-binding peptide, TFPI-inhibitory peptide)of the invention. Such a nucleic acid, in one aspect, is providedinstead of, or in addition to, a peptide complex or peptide (e.g.,TFPI-binding, TFPI-inhibitory peptide). Expression vectors, nucleic acidregulatory sequences, administration methods, and the like, are furtherdescribed herein and in U.S. Patent Publication No. 20030045498.

A particular administration regimen for a particular subject willdepend, in part, upon the TFPI-inhibitory peptide of the invention used,the amount of TFPI-binding peptide (e.g., TFPI-inhibitory peptide)administered, the route of administration, the particular ailment beingtreated, considerations relevant to the recipient, and the cause andextent of any side effects. The amount of peptide administered to asubject (e.g., a mammal, such as a human) and the conditions ofadministration (e.g., timing of administration, route of administration,dosage regimen) are sufficient to affect the desired biological responseover a reasonable time frame. Dosage typically depends upon a variety offactors, including the particular TFPI-binding peptide (e.g.,TFPI-inhibitory peptide) employed, the age and body weight of thesubject, as well as the existence and severity of any disease ordisorder in the subject. The size of the dose also will be determined bythe route, timing, and frequency of administration. Accordingly, theclinician may titer the dosage and modify the route of administration toobtain the optimal therapeutic effect, and conventional range-findingtechniques are known to those of ordinary skill in the art. Purely byway of illustration, in one aspect, the method comprises administering,e.g., from about 0.1 μg/kg to about 100 mg/kg or more, depending on thefactors mentioned above. In other embodiments, the dosage may range from1 μg/kg up to about 75 mg/kg; or 5 μg/kg up to about 50 mg/kg; or 10μg/kg up to about 20 mg/kg. In certain embodiments, the dose comprisesabout 0.5 mg/kg to about 20 mg/kg (e.g., about 1 mg/kg, 1.5 mg/kg, 2mg/kg, 2.3 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, or 10mg/kg) of peptide. Given the chronic nature of many blood coagulationdisorders, it is envisioned that a subject will receive the TFPI-bindingpeptide (e.g., TFPI-inhibitory peptide) over a treatment course lastingweeks, months, or years, and may require one or more doses daily orweekly. In other embodiments, the TFPI-binding peptide (e.g.,TFPI-inhibitory peptide) is administered to treat an acute condition(e.g., bleeding caused by surgery or trauma, or factorinhibitor/autoimmune episodes in subjects receiving coagulationreplacement therapy) for a relatively short treatment period, e.g., oneto 14 days.

Suitable methods of administering a physiologically-acceptablecomposition, such as a pharmaceutical composition comprising a peptidedescribed herein, are well known in the art. Although more than oneroute can be used to administer a peptide, a particular route canprovide a more immediate and more effective reaction than another route.Depending on the circumstances, a pharmaceutical composition is appliedor instilled into body cavities, absorbed through the skin or mucousmembranes, ingested, inhaled, and/or introduced into circulation. In oneaspect, a composition comprising a TFPI-binding peptide (e.g.,TFPI-inhibitory peptide) is administered intravenously, intraarterially,or intraperitoneally to introduce the peptide of the invention intocirculation. Non-intravenous administration also is appropriate,particularly with respect to low molecular weight therapeutics. Incertain circumstances, it is desirable to deliver a pharmaceuticalcomposition comprising the TFPI-binding peptide (e.g., TFPI-inhibitorypeptide) orally, topically, sublingually, vaginally, rectally,pulmonary; through injection by intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraportal,intralesional, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intranasal, urethral, or enteral means; bysustained release systems; or by implantation devices. If desired, theTFPI-binding peptide (e.g., TFPI-inhibitory peptide) is administeredregionally via intraarterial or intravenous administration feeding aregion of interest, e.g., via the femoral artery for delivery to theleg. In one embodiment, the peptide is incorporated into a microparticleas described in, for example, U.S. Pat. Nos. 5,439,686 and 5,498,421,and U.S. Patent Publications 2003/0059474, 2003/0064033, 2004/0043077,2005/0048127, 2005/0170005, 2005/0142205, 2005/142201, 2005/0233945,2005/0147689. 2005/0142206, 2006/0024379, 2006/0260777, 2007/0207210,2007/0092452, 2007/0281031, and 2008/0026068. Alternatively, thecomposition is administered via implantation of a membrane, sponge, oranother appropriate material on to which the desired molecule has beenabsorbed or encapsulated. Where an implantation device is used, thedevice in one aspect is implanted into any suitable tissue, and deliveryof the desired molecule is in various aspects via diffusion,timed-release bolus, or continuous administration. In other aspects, theTFPI-inhibitory peptide is administered directly to exposed tissueduring surgical procedures or treatment of injury, or is administeredvia transfusion of blood procedures. Therapeutic delivery approaches arewell known to the skilled artisan, some of which are further described,for example, in U.S. Pat. No. 5,399,363.

To facilitate administration, the TFPI-binding peptide (e.g.,TFPI-inhibitory peptide) or peptide complex in one embodiment isformulated into a physiologically-acceptable composition comprising acarrier (i.e., vehicle, adjuvant, buffer, or diluent). The particularcarrier employed is limited only by chemico-physical considerations,such as solubility and lack of reactivity with the peptide, and by theroute of administration. Physiologically-acceptable carriers are wellknown in the art. Illustrative pharmaceutical forms suitable forinjectable use include without limitation sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions (for example, see U.S. Pat.No. 5,466,468). Injectable formulations are further described in, e.g.,Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia.Pa., Banker and Chalmers. eds., pages 238-250 (1982), and ASHP Handbookon Injectable Drugs, Toissel, 4^(th) ed., pages 622-630 (1986)). Apharmaceutical composition comprising a peptide provided herein isoptionally placed within containers, along with packaging material thatprovides instructions regarding the use of such pharmaceuticalcompositions. Generally, such instructions include a tangible expressiondescribing the reagent concentration, as well as, in certainembodiments, relative amounts of excipient ingredients or diluents thatmay be necessary to reconstitute the pharmaceutical composition.

When appropriate, the TFPI-binding peptide (e.g., TFPI-inhibitorypeptide) or peptide complex of the invention is administered incombination with other substances and/or other therapeutic modalities toachieve an additional or augmented biological effect. Co-treatmentsinclude, but are not limited to, plasma-derived or recombinantcoagulation factors, hemophilia prophylaxis treatments,immunosuppressants, plasma factor-inhibiting antibody antagonists (i.e.,anti-inhibitors), antifibrinolytics, antibiotics, hormone therapy,anti-inflammatory agents (e.g., Non-Steroidal Anti-Inflammatory Drugs(NSAIDs) or steroidal anti-inflammatory substances), procoagulants, andpain relievers. In one aspect, the method is an adjunct therapy totraditional replacement factor treatment regimens involvingadministration of, e.g., FXIII, FXII, FXI (e.g., HEMOLEVEN® (Laboratoirefrancais du Fractionnement et des Biotechnologies, Les Ulis, France) andFXI concentrate (BioProducts Laboratory, Elstree, Hertfordshire, UK)),FX, FIX (e.g., BENEFIX® Coagulation Factor IX (Wyeth, Madison, N.J.);ALPHANINE® SD (Grifols, Los Angeles, Calif.); MONONINE® (CSL Behring,King of Prussia, Pa.); BEBULIN-VH™ (Baxter, Deerfield, Ill.);PROFILNINE® SD (Grifols, Los Angeles, Calif.); or PROPLEX T™ (Baxter,Deerfield, Ill.)), FVIII (e.g., ADVATE™ (Baxter, Deerfield, Ill.);HELIXATE® FS(CSL Behring, King of Prussia, Pa.); REFACTO® (Wyeth,Madison, N.J.), XYNTHA™ (Wyeth, Madison, N.J.), KOGENATE® and KOGENATE®FS (Bayer, Pittsburgh, Pa.); ALPHANATE® (Grifols, Los Angeles, Calif.);HEMOPHIL M™ (Baxter, Deerfield, Ill.); KOATE®-DVI (TalecrisBiotherapeutics-USA, Research Triangle Park, N.C.); or MONARC-M™(Baxter, Deerfield, Ill.)), FVIIa (e.g., NOVOSEVEN® FVIIa (Novo Nordisk,Princeton, N.J.) and FVII concentrate (Baxter Bioscience, Vienna,Austria, or BioProducts Laboratory, Elstree, Hertfordshire, UK)), FV,FVa, FII, and/or FIII, to a subject. In some instances, the subject alsoreceives FEIBA VH Immuno™ (Baxter BioScience, Vienna, Austria), which isa freeze-dried sterile human plasma fraction with Factor VIII inhibitorbypassing activity. FEIBA VH Immuno™ contains approximately equal unitsof Factor VIII inhibitor bypassing activity and Prothrombin ComplexFactors (Factors II, VII, IX, and X and protein C). Other exemplaryco-treatments include, but are not limited to, prekallikrein, highmolecular weight kininogen (HMWK), Von Willebrand's factor, TissueFactor, and thrombin. Alternatively or in addition, the TFPI-bindingpeptide (e.g., TFPI-inhibitory peptide) is co-formulated with one ormore different TFPI-binding peptides (e.g., TFPI-inhibitory peptides).In one aspect, administration of the TFPI-binding peptide allows areduction in the dose of co-therapeutic required to achieve a desiredbiological response.

The invention thus includes administering to a subject a TFPI-bindingpeptide (e.g., TFPI-inhibitory peptide) of the invention (or multipleTFPI-binding peptides, or a peptide complex), in combination with one ormore additionally suitable substances(s), each being administeredaccording to a regimen suitable for that medicament. Administrationstrategies include concurrent administration (i.e., substantiallysimultaneous administration) and non-concurrent administration (i.e.,administration at different times, in any order, whether overlapping ornot) of the TFPI-binding peptide (e.g., TFPI-inhibitory peptide) (orpeptide complex) and one or more additionally suitable agents(s). Itwill be appreciated that different components are optionallyadministered in the same or in separate compositions, and by the same ordifferent routes of administration.

In some embodiments, the peptide of the invention is conjugated to amoiety, e.g., a therapeutic or diagnostic moiety, such as the detectionmoieties and co-treatments described above. Alternatively or inaddition, the peptide is administered in combination with an interactionpartner (e.g., an antibody, antibody fragment, anticalin, aptamer, orspiegelmer) that (a) binds the peptide and (b) is therapeutically activeand/or is linked to a moiety that provides additional functionality tothe interaction partner (e.g., a therapeutic, diagnostic, or detectionagent). Suitable moieties include, but are not limited to,photosensitizers, dyes, radionuclides, radionuclide-containingcomplexes, enzymes, toxins, antibodies, antibody fragments, andcytotoxic agents, and, in some instances, the moiety possessestherapeutic activity (i.e., achieves an advantageous or desiredbiological effect). The peptide conjugates or peptide-interactionpartner pair is suitable for use in any of the methods described herein,such as methods of treating a subject suffering from a disease ordisorder or at risk of suffering from a disease or disorder.

The invention further provides a method for inhibiting degradation ofTFPI by a serine protease. Protease degradation can complicate handlingof TFPI in research settings. Additionally, although TFPI inhibition isdesired to improve various medical conditions (e.g., hemophilia), TFPImay be desired in other clinical embodiments to, for example, reducecoagulation. By “inhibiting” is meant protection, in whole in part, fromdegradation or cleavage by a protease. Serine proteases are wellcharacterized and include, e.g., elastase, thrombin, plasmin, FXa, orchymase. The method comprises contacting TFPI with a peptide comprisingthe structure of formula (XIV):

X7001-X7002-X7003-X7004-X7005-X7006-(XIV) (SEQ ID NO: 3154),

wherein X7001 is either present or absent, whereby in case X7001 ispresent it is an amino acid selected from the group consisting of A, C,C(NEM), D, E, F, G, H, I, K, L, P, R, S, T, V and W;

wherein X7002 is either present or absent, whereby in case X7002 ispresent it is an amino acid selected from the group consisting of A, C,C(NEM), D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y;

wherein X7003 is an amino acid selected from the group consisting of A,F, I, K, L, R, S, T, V, W and Y;

wherein X7004 is an amino acid selected from the group consisting of A,D, E, F, G, I, K, L, R, S, T, V and W;

wherein X7005 is R or W;

wherein X7006 is an amino acid selected from the group consisting of F,H, I, K, L, R, V and W;

wherein X7007 is an amino acid selected from the group consisting ofOrn, homoK, C, Hcy, Dap and K, preferably selected from the groupconsisting of C and Hcy;

wherein X7008 is an amino acid selected from the group consisting of A,G, R, S and T;

wherein X7009 is an amino acid selected from the group consisting of a,A, I, K, L, M, m, Moo, Nle, p, R, Sem and V;

wherein X7010 is an amino acid selected from the group consisting of A,G, I, K, L, P, R, S, T and V;

wherein X7011 is an amino acid selected from the group consisting of D,E, G, S and T;

wherein X7012 is an amino acid selected from the group consisting of A,a, D, d, E, e, F, f, G, I, K, k, L, l, M, m, Moo, Nle, nle, P, p, R, r,S, s, Sem, T, t, V, v, W and w;

wherein X7013 is an amino acid selected from the group consisting of A,C, C(NEM), Con, Con(Meox), D, d, E, e, Eag, F, G, I, K, L, N, R, S, s,T, V and W;

wherein X7014 is an amino acid selected from the group consisting of A,D, E, F, G, I, K, L, M, R, S, T, V and W;

wherein X7015 is an amino acid selected from the group consisting of A,D, E, F, G, I, K, L, M, Nle, R, S, T, V and W;

wherein X7016 is an amino acid selected from the group consisting of A,D, E, F, I, K, L, M, Moo, Nle, R, S, Sem, T, V, W and Y;

wherein X7017 is an amino acid selected from the group consisting of A,D, E, F, G, I, K, L, R, S, T, V, W and Y;

wherein X7018 is an amino acid selected from the group consisting of Cand D, preferably C;

wherein X7019 is an amino acid selected from the group consisting of A,F, I, L, S, T, V and W;

wherein X7020 is an amino acid selected from the group consisting of Fand W;

wherein X7021 is an amino acid selected from the group consisting of I,L and V;

wherein X7022 is an amino acid selected from the group consisting of A,D, E, F, G, I, K, L, P, R, S, T, V and W;

wherein X7023 is either present or absent, whereby in case X7023 ispresent it is an amino acid selected from the group consisting of A, C,C(NEM), Con, Con(Meox), D, E, Eag, F, G, I, K, L, R, S, T, V, W and Y;and

wherein the peptide comprises as a cyclic structure generated by alinkage between X7007 and X7018, whereby degradation of TFPI by theserine protease is inhibited. Optionally, X7001 is an amino acidselected from the group consisting of A, D, F, G, H, K, L and S; X7002is an amino acid selected from the group consisting of H, F, M and R;X7003 is an amino acid selected from the group consisting of F and Y;X7004 is K; X7005 is W; X7006 is an amino acid selected from the groupconsisting of F and H; X7007 is C; X7008 is an amino acid selected fromthe group consisting of A, G and S; X7009 is an amino acid selected fromthe group consisting of M, Sem and V; X7010 is an amino acid selectedfrom the group consisting of K, P and R; X7011 is D; wherein X7012 is anamino acid selected from the group consisting of F, L, l, M and Sem;wherein X7013 is an amino acid selected from the group consisting of D,G, K and S; wherein X7014 is G; wherein X7015 is an amino acid selectedfrom the group consisting of I and T; wherein X7016 is an amino acidselected from the group consisting of D, F, M, Sem and Y; wherein X7017is an amino acid selected from the group consisting of S and T; whereinX7018 is C; wherein X7019 is an amino acid selected from the groupconsisting of A and V; wherein X7020 is W; wherein X7021 is V; whereinX7022 is an amino acid selected from the group consisting of F, L, K, R,P and W; wherein X7023 is either present or absent, whereby in caseX7023 is present it is an amino acid sequence selected from the groupconsisting of A, D, F, M, S and Y; and wherein the peptide comprises asa cyclic structure generated by a linkage between X7007 and X7018. The“contacting” may be performed in vitro or in vivo. The dosage and routeof administration considerations described herein are applicable to themethod of inhibiting proteolytic degradation.

The peptide is optionally part of a peptide complex that furthercomprises a peptide comprising the structure of formula (XIII):

X6001-X6002-X6003-X6004-X6005-X6006-X6007-X6008-X6009-X6010-X6011-X6012-X6013-X6014-X6015-X6016-X6017-X6018-X6019-X6020(XIII) (SEQ ID NO: 3153);

wherein X6001 is an amino acid selected from the group consisting of F,L, M, Y, 1Ni, Thi, Bta, Dopa, Bhf, C, D, G, H, I, K, N, Nmf, Q, R, T, V,and W;

wherein X6002 is an amino acid selected from the group consisting of Q,G, and K;

wherein X6003 is an amino acid selected from the group consisting of C,D, E, M, Q, R, S, T, Ede(O), Cmc, A, Aib, Bhs, F, G, H, I, K, L, N, P,V, W and Y;

wherein X6004 is an amino acid selected from the group consisting ofAib, E, G, I, K, L, M, P, R, W, Y, A, Bhk, C, D, F, H, k, N, Nmk, Q, S,T and V;

wherein X6005 is an amino acid selected from the group consisting of a,A, Aib, C, D, d, E, G, H, K, k, M, N, Nmg, p, Q, R, NpropylG, aze, pip,tic, oic, hyp, nma, Ncg, Abg, Apg, thz, dtc, Bal, F, L, S, T, V, W andY;

wherein X6006 is an amino acid selected from the group consisting of A,C, C(NEM), D, E, G, H, K, M, N, Q, R, S, V, Cit, C(Acm), Nle, I, Ede(O),Cmc, Ed, Eea, Eec, Eef, Nif, Eew, Aib, Btq, F, I, L, T, W and Y;

wherein X6007 is an amino acid selected from the group consisting of I,V, T, Chg, Phg, Tle, A, F, G, I, K, L, Nmv, P, Q, S, W and Y;

wherein X6008 is an amino acid selected from the group consisting of F,H, 1Ni, 2Ni, Pmy, Y, and W;

wherein X6009 is an amino acid selected from the group consisting ofAib, V, Chg, Phg, Abu, Cpg, Tle, L-2-amino-4,4,4-trifluorobutyric acid,A, f, I, K, S, T and V;

wherein X6010 is an amino acid selected from the group consisting of A,C, D, d, E, F, H, K, M, N, P, Q, R, S, T, V, W, Y, Nmd, C(NEM), Aib, G,I, L and Nmf;

wherein X6011 is an amino acid selected from the group consisting of A,a, G, p, Sar, c, hcy, Aib, C, K, G and Nmg;

wherein X6012 is an amino acid selected from the group consisting of Y,Tym, Pty, Dopa and Pmy;

wherein X6013 is an amino acid selected from the group consisting ofAib, C, F, 1Ni, Thi, Bta, A, E, G, H, K, L, M, Q, R, W and Y;

wherein X6014 is an amino acid selected from the group consisting of A,Aib, C, C(NEM), D, E, K, L, M, N, Q, R, T, V, Hcy, Bhe, F, G, H, I, P,S, W and Y;

wherein X6015 is an amino acid selected from the group consisting of R,(omega-methyl)-R, D, E and K;

wherein X6016 is an amino acid selected from the group consisting of L,Hcy, Hle and Aml;

wherein X6017 is an amino acid selected from the group consisting of A,a, Aib, C, c, Cha, Dab, Eag, Eew, H, Har, Hci, Hle, I, K, L, M, Nle,Nva, Opa, Orn, R, S, Deg, Ebc, Eca, Egz, Aic, Apc, Egt,(omega-methyl)-R, Bhr, Cit, D, Dap, E, F, G, N, Q, T, V, W and Y;

wherein X6018 is an amino acid selected from the group consisting of A,Aib, Hcy, hcy, C, c, L, Nle, M, N, R, Bal, D, E, F, G, H, I, K, Q, S, T,V, W and Y;

wherein X6019 is an amino acid selected from the group consisting of K,R, Har, Bhk and V; and

wherein X6020 is an amino acid selected from the group consisting of K,L, Hcy, Aml, Aib, Bhl, C, F, G, H, I, Nml, Q, R, S, T, V, W and Y.

The invention further provides a method for identifying a TFPI-bindingcompound, such as a TFPI-binding peptide. In one aspect, the methodcomprises (a) contacting a peptide comprising TFPI Kunitz domain 1 (KD1)with a TFPI-binding peptide described herein and a test compound underconditions that allow formation of KD1-TFPI-binding peptide complexes.The method further comprises (b) measuring KD1-TFPI-binding peptidecomplexes formed in step (a), and (c) comparing the number ofKD1-TFPI-binding peptide complexes formed in the presence of the testcompound with the number of KD1-TFPI-binding peptide complexes formed inthe absence of the test compound. A reduction in the number ofKD1-TFPI-binding peptide complexes formed in the presence of the testcompound compared to the number of KD1-TFPI-binding peptide complexesformed in the absence of the test compound indicates that the testcompound is a TFPI-binding compound. In one aspect, the method furthercomprises forming KD1-TFPI-binding complexes in the absence of the testcompound for comparison in step (c), although this is not requiredinasmuch as the information may be obtained separately (e.g., frompreviously prepared reference standards).

KD1, the TFPI-binding peptide, and the test compound are combinedsimultaneously or sequentially, optionally with washing steps beforeand/or after addition of the TFPI-binding peptide and/or the testcompound. In one embodiment, the peptide comprising KD1 is contactedwith a TFPI-binding peptide described herein under conditions that allowformation of KD1-TFPI-binding peptide complexes, unbound TFPI-bindingpeptide is removed, and the remaining KD-peptide complexes are contactedwith a test compound. Displacement of the TFPI-binding peptide from theTFPI-peptide complexes is detected, and indicates that the test compoundis a TFPI-binding compound. Displacement is detected by, for example,measuring the number of KD1-TFPI-binding peptide complexes before andafter exposure to the test compound.

KD1-TFPI-binding peptide complexes are detected and/or measured(quantified) using any suitable detection means, including detectionmeans known in the art for detecting peptides in a sample. For example,in one embodiment of the invention, the TFPI-binding peptide comprises alabel that generates a signal. Exemplary labels are described herein andinclude, e.g., radionuclides, fluorescent dyes, isotopes, enzymesubstrates, and enzymes. The method comprises measuring signal generatedby KD1-TFPI-binding peptide complexes and comparing signal generated byKD1-TFPI-binding peptide complexes formed in the presence of the testcompound with signal generated by KD1-TFPI-binding peptide complexesformed in the absence of the test compound. A reduction in signal from asample comprising KD1-TFPI-binding peptide complexes exposed to testcompound (compared to signal generated by a similar sample ofKD1-TFPI-binding peptide complexes not exposed to the test compound)indicates that complex formation has been inhibited or disrupted, andthat the test compound is a TFPI-binding compound.

The invention also provides a method of identifying a TFPI-bindingcompound that interferes with TFPI-FXa interactions. The method ispredicated, at least in part, on the surprising discovery that TFPI KD1binds to an exosite of FXa and contributes to TFPI's inhibition of FXaactivity. In one aspect, the method comprises contacting a peptideconsisting essentially of KD1 (i.e., a peptide comprising KD1 in theabsence of KD2) with FXa in the presence of a test compound underconditions that allow binding of KD1 to FXa. The method furthercomprises comparing KD1-FXa binding in the presence of the test compoundwith KD1-FXa binding in the absence of the test compound. A decrease inKD1-FXa binding in the presence of the test compound compared to KD1-FXabinding in the absence of the test compound indicates that the testcompound is a TFPI-binding compound. KD1-FXa binding can be detectedand/or quantitated using any method, such as the methods describedherein. For example, KD1 or FXa is labeled, and the signal generated byKD1-FXa complexes exposed to the test compound is compared to the signalgenerated by KD1-FXa complexes not exposed to the test compound.

The methods of the invention to identify TFPI-binding compounds areparticularly amenable to the various high throughput screeningtechniques known in the art. Any “test compound” (e.g., small molecule,peptide, protein (such as an antibody or fragment thereof),peptidomimetic, or polynucleotide (DNA or RNA)) is suitable forscreening using the methods described herein. If desired, a collection,population, or library of test compounds is screened for TFPI binding(and, optionally, anti-TFPI activity) using the methods describedherein. There are a number of different libraries used for theidentification of TFPI inhibitors, including, but not limited to,chemical libraries, natural product libraries, and combinatoriallibraries comprising peptides and/or organic molecules. A chemicallibrary, in some aspects, consists of structural analogs of knowncompounds or compounds that are identified as “hits” or “leads” viaother screening methods. Natural product libraries are collections ofsubstances isolated from or produced by microorganisms, animals, plants,or marine organisms. Combinatorial libraries are composed of largenumbers of peptides or organic compounds, typically as a mixture. Themethods described herein also are useful for screening a display ornucleic acid library, such as a yeast display library, a bacterialdisplay library, a phage display library, a ribosome display library, anmRNA display library, a RNA library, or a DNA library. One method ofscreening a display library is exemplified in Example 1. High throughputscreening methods embraced by the invention include automated proceduresallowing screening of tens to hundreds of thousands of test compounds.

In another aspect, the inventive method for identifying a TFPI-bindingcompound comprises contacting a peptide comprising (or consisting of)KD1 with a test compound, and detecting binding of the test compound toa TFPI binding site defined by KD1 amino acid residues corresponding tohuman TFPI residues Phe28, Lys29, Ala30, Asp32, Ile46, Phe47, and Ile55,such as a binding site defined by human TFPI residues Ala27, Phe28,Lys29, Ala30, Asp31, Asp32, Lys36, Ile38, Ile46, Phe47, and Ile55. Inone embodiment, the binding site is defined by amino acid residuescorresponding to human TFPI residues Ala27, Phe28, Lys29, Ala30, Asp31,Asp32, Lys36, Ala37, Ile38, Phe44, Ile46, Phe47, and Ile55. The bindingsite corresponds to the TFPI binding site of JBT1857, a TFPI-bindingpeptide that inhibits TFPI activity in a number of functional assays.JBT1857 is an example of a peptide of the JBT0047 class of peptidesrepresented herein as, e.g., formulas (I)-(IV) and (XI), and examples ofwhich are set forth in FIGS. 32, 62, and 65.

Additionally, the invention provides a method for identifying aTFPI-binding compound comprises contacting a peptide comprising KD1-KD2(including the region of the TFPI polypeptide linking KD1 and KD2) witha test compound, and detecting binding of the test compound to a TFPIbinding site defined by KD1-KD2 amino acid residues corresponding tohuman TFPI residues R41, Y53, C59, E60, Q63, R65, E67, E71, K74, M75,N80, N82, R83, I84, I85, T87, F96, C106, C130, L131, N133, M134, N136,F137, E142, N145, and I146. The binding site corresponds to the TFPIbinding site of JBT1837, a TFPI-binding peptide that inhibits TFPIactivity in a number of functional assays. JBT1837 is an example of apeptide of the JBT0120 class of peptides represented herein as, e.g.,formulas (V)-(VII), and examples of which are set forth in FIG. 34.

The TFPI binding site amino acid residues described herein are inreference to the human TFPI amino acid sequence, and the numberingrefers to the position of the recited amino acid in relation to theN-terminus of human TFPI. Merely for the purpose of illustrating theposition of the TFPI binding site, the amino acid sequence of a fragmentof human TFPI comprising KD1 is provided as SEQ ID NO: 4234(DSEEDEEHTIITDTELPPLKLMHSFCAFKADDGPCKAIMKRFFFNIFTRQCEEFIGGCEGNQNRFESLEECKKMCTRDNA (amino acids 26-75 encoding KD1 are indicated inbold)). Corresponding amino acids of other TFPI polypeptides (such asTFPI polypeptides from different organisms, or TFPI polypeptidefragments) are identified, for example, by aligning a polypeptide'samino acid sequence with SEQ ID NO: 4234. While, in one embodiment, thepeptide comprising TFPI KD1 does not comprise other regions of the TFPIprotein responsible for TFPI activity, other embodiments entail the useof a peptide comprising amino acids 1-160 of human TFPI (comprising KD1and KD2) or comprising full length human TFPI (containing KD1-KD3).

Binding of a test compound to the TFPI binding site defined herein isdetected using any of a number methods, including the detection methodsdescribed herein. An exemplary method for detecting binding employsnuclear magnetic resonance (NMR) to recognize chemical shifts at aminoacid residues within the TFPI binding site. Chemical shifts at TFPIamino acid positions 28-30, 32, 46, 47, and 55, and optionally positions27, 31, 36-38, and 44, denotes interaction of the test compound withthese amino acid contact points on TFPI. Similarly, chemical shifts atTFPI amino acid positions 41, 53, 59, 60, 96, 106, 130, 133, 136, 137,142, 63, 65, 67, 71, 74, 75, 80, 82-85, 87, 131, 134, 145, and 146denotes interaction of a test compound with these amino acid contactpoints on TFPI. To determine the presence or absence of chemical shiftsat particular amino acids resulting from test compound binding, NMR dataobtained from the KD1-test compound complex is compared to NMR dataobtained from free TFPI peptide (e.g., free KD1 peptide or free KD1-KD2peptide). Use of NMR to detect binding between a test compound and TFPIis further described in the Examples.

Alternatively, binding of a test compound to the TFPI-binding sitedefined herein is determined indirectly by detecting alterations in theability of TFPI KD1, optionally in combination with KD2, to interactwith its natural binding partners, e.g., FVIIa or FXa. In this regard,the method, in one aspect, comprises contacting the peptide comprisingTFPI KD1 with FVIIa in the presence of the test compound underconditions that allow binding of KD1 to FVIIa, and KD1-FVIIa binding iscompared with KD1-FVIIa binding in the absence of the test compound.Alternatively or in addition, the method comprises contacting thepeptide comprising TFPI KD1 with FXa in the presence of the testcompound under conditions that allow binding of KD1 to FXa, andcomparing KD1-FXa binding in the presence of the test compound withKD1-FXa binding in the absence of the test compound. Optionally, thepeptide comprising KD1 also comprises KD2, and the method comprisescontacting the peptide with FXa in the presence of a test compound underconditions that allow binding of KD2 to FXa, and KD2-FXa binding iscompared with KD2-FXa binding in the absence of the test compound. Adecrease in KD1-FVIIa binding, KD1-FXa binding, or KD2-FXa binding inthe presence of the test compound (compared to KD1-FVIIa binding,KD1-FXa binding, or KD2-FXa binding in the absence of the test compound)indicates that the test compound is a TFPI-binding compound. The methodoptionally comprises contacting KD1 and/or KD2 to FVIIa and/or FXa inthe absence of the test compound as a reference for comparing binding inthe presence of the test compound.

KD binding to FVIIa or FXa is determined and/or quantified using anysuitable method for detecting protein-protein interactions, such as themethods described herein using detectable labels. Binding of the testcompound to the TFPI binding site is, alternatively, detected using anenzymatic assay. FVIIa or FXa enzymatic activity is a suitable surrogatefor evaluating binding of the proteins to TFPI KD1 or KD2; testcompounds that bind the TFPI-binding site defined herein inhibit TFPIactivity, resulting in increased FVIIa and FXa activity. Enzymaticassays for evaluating FVIIa or FXa activity are described in detailherein.

The invention further includes compounds identified as TFPI-bindingcompounds in the methods of the invention, as well as compositionscomprising one or more identified compounds. Methods for isolating orpurifying a compound, such as TFPI-binding compound (e.g., aTFPI-binding peptide) identified as described herein are known in theart and described above. In some aspects, TFPI-binding compoundsidentified as described herein are TFPI inhibitors that downregulate orablate one or more TFPI activities. The invention provides a TFPIinhibitor that binds human TFPI at a first binding site defined by aminoacid residues F28, K29, A30, D32, I46, F47, and I55 (e.g., a bindingsite defined by amino acid residues A27, F28, K29, A30, D31, D32, K36,I38, I46, F47, and I55, such as a binding site defined by amino acidresidues A27, F28, K29, A30, D31, D32, K36, A37, I38, F44, I46, F47, andI55) and a second binding site defined by amino acid residues R41, Y53,C59, E60, Q63, R65, E67, E71, K74, M75, N80, N82, R83, I84, I85, T87,F96, C106, C130, L131, N133, M134, N136, F137, E142, N145, and I146. Theamino acid designations correspond to amino acid residue positions inhuman TFPI. In various aspects, the TFPI inhibitor is a peptide. TheTFPI inhibitor, in various embodiments, comprises a first peptide and asecond peptide linked by a linker moiety as described herein.

In one embodiment, the invention includes a method for purifying acompound that inhibits FXa activity. The method comprises contacting apeptide comprising TFPI KD1 with a compound under conditions that allowformation of compound-KD1 complexes, removing unbound compound, anddissociating the compound-KD1 complexes to release the compound, whichbinds TFPI. Use of a TFPI inhibitor identified and/or purified asdescribed herein for the manufacture of a medicament, such as amedicament for treating a blood coagulation disorder, is provided, aswell as a method for treating a subject suffering from a disease or atrisk of suffering from a disease comprising administering the TFPIinhibitor to the subject.

In addition, a method of inhibiting human TFPI is provided, wherein themethod comprises contacting human TFPI with an inhibitor that bindshuman TFPI at a binding site defined by amino acid residues Phe28,Lys29, Ala30, Asp32, Ile46, Phe47, and Ile55. Another aspect of theinvention includes a method for treating a subject suffering from adisease or at risk of suffering from a disease. The method comprisesadministering to the subject an inhibitor that binds human TFPI at abinding site defined by amino acid residues Phe28, Lys29, Ala30, Asp32,Ile46, Phe47, and Ile55. In one aspect, the human TFPI binding site isdefined by amino acid residues Ala27, Phe28, Lys29, Ala30, Asp31, Asp32,Lys36, Ile38, Ile46, Phe47, and Ile55, such as a binding site defined byamino acid residues Ala27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36,Ala37, Ile38, Phe44, Ile46, Phe47, and Ile55. Alternatively or inaddition, the method of inhibiting TFPI comprises contacting human TFPIwith an inhibitor that binds human TFPI at a binding site defined byamino acid residues R41, Y53, C59, E60, Q63, R65, E67, E71, K74, M75,N80, N82, R83, I84, I85, T87, F96, C106, C130, L131, N133, M134, N136,F137, E142, N145, and I146. The invention further provides a method oftreating a subject suffering from, or at risk of suffering from, adisease, the method comprising administering to a subject an inhibitorthat binds human TFPI at a binding site defined by amino acid residuesR41, Y53, C59, E60, Q63, R65, E67, E71, K74, M75, N80, N82, R83, I84,I85, T87, F96, C106, C130, L131, N133, M134, N136, F137, E142, N145, andI146. In various aspects, the inhibitor binds TFPI at a binding sitedefined by amino acid residues F28, K29, A30, D32, I46, F47, and I55(and optionally A27, D31, K36, A37, I38, and F44) and a binding sitedefined by amino acid residues R41, Y53, C59, E60, F96, C106, C130,N133, N136, F137, E142, Q63, R65, E67, E71, K74, M75, N80, N82, R83,I84, I85, T87, L131, M134, N145, and I146.

Any inhibitor that contacts the TFPI binding site(s) defined herein andinhibits (downregulates or ablates) one or more TFPI activity issuitable for use in the context of the method. The TFPI inhibitor is,optionally, a TFPI-binding peptide, such as a TFPI-binding peptidehaving the characteristics described herein.

The invention further includes computer storage media and methods formodeling candidate TFPI-compounds in the TFPI binding site definedherein. Three dimensional (3D) modeling of proteins can be used inconjunction with 3D models of various test TFPI-binding compounds (e.g.,peptides or small molecules) to determine fit between the compounds andtargeted amino acids in TFPI. Because the effectiveness of a testcompound in inhibiting TFPI can be limited if the compound does notremain attached to TFPI for a sufficient period of time to effect abiological response, the tendency of the two to remain coupled can bepredicted to develop an affinity rating.

By analyzing the 3D surface of the TFPI protein and the fit of thecorresponding compound to the surface in view of the affinity rating,modifications to the compound (e.g., peptide) can be developed toimprove both the number of contact points between the surface and thecompound and the strength of the bonds at the contact points. Theeffectiveness of chemical-based candidates and peptide-based TFPIinhibitors can similarly be modeled using this technique, whichfacilitates the rational design of TFPI-binding compounds. A computermodel of the three dimensional (3D) surface of KD1 (optionally linked toKD2) allows testing of the ability of various peptides or chemicals toattach to an identified subset of amino acids that define a TFPI bindingsite and inhibit KD1 (and, optionally, KD2). In one aspect, a surface ofthe KD1 protein is modeled in 3D space on a computer, particularly asurface bounded by the targeted amino acids in KD1. The 3D models ofvarious peptides, for example, can be matched to the surface todetermine how many of the target TFPI amino acids are contacted by thepeptide and also to develop an affinity rating predicting how long thepeptide will remain attached to the target surface.

By changing the peptide model and repeating the computer modeling,affinity ratings can be quickly generated for a peptide family. The mostpromising peptide variants (e.g., a second peptide comprising one ormore substitutions within the amino acid sequence of a parent peptide)can be singled out for further physical testing, if desired.

The invention provides a computer storage media having computerexecutable instructions that, when executed on the processor of acomputer, implement a method of modeling interaction between selectedthree dimensional (3D) points in a TFPI KD1 protein and a test compound.The method comprises obtaining a protein structure 3D model for the TFPIKD1 protein; determining a 3D relationship between a selected subset ofamino acids in the protein structure, wherein the selected subset ofamino acids comprises Phe28, Lys29, Ala30, Asp32, Ile46, Phe47, andIle55; modeling a surface bounded by the selected subset of amino acids;obtaining a test compound 3D model of a test compound; matching the testcompound 3D model to the surface bounded by the selected subset of aminoacids; and identifying contact points between the selected subset ofamino acids of the surface and the test compound 3D model. Optionally,the method further comprises determining a number of the contact pointsbetween the surface and the test compound 3D model; and recording anaffinity rating for the test compound 3D model corresponding to thenumber of contact points. In one aspect, the selected subset of aminoacids comprises (or consists of) Ala27, Phe28, Lys29, Ala30, Asp31,Asp32, Lys36, Ala37, Ile38, Phe44, Ile46, Phe47, and Ile55. The methodfurther optionally comprises obtaining an updated test compound 3D modelbased on a second test compound; matching the updated test compound 3Dmodel to the surface bounded by the selected subset of amino acids; andidentifying the identified contact points between the selected subset ofamino acids of the surface and the updated test compound 3D model on adisplay of the computer. In one embodiment, the method further comprisesdetermining a number of the contact points between the surface and theupdated test compound 3D model; determining a bond type for each contactpoint between the surface and the updated test compound 3D model; andrecording a new affinity rating based on the number of contact pointsand an aggregate of the bond types for each contact point between thesurface and the updated test compound 3D model. The updated affinityrating is then compared with the new affinity rating to determinewhether the test compound or the second test compound has a higheraffinity rating, if desired. The contact points can be displayed on thecomputer, thereby facilitating optimization or design of TFPI-bindingcompounds.

Additionally, the invention provides a computer storage media havingcomputer executable instructions that, when executed on the processor ofa computer, implement a method of modeling interaction between selectedthree dimensional (3D) points in a TFPI KD1-KD2 protein and a testcompound. The method comprises obtaining a protein structure 3D modelfor the TFPI KD1-KD2 protein region; determining a 3D relationshipbetween a selected subset of amino acids in the protein structure,wherein the selected subset of amino acids comprises R41, Y53, C59, E60,F96, C106, C130, N133, N136, F137, E142, Q63, R65, E67, E71, K74, M75,N80, N82, R83, I84, I85, T87, L131, M134, N145, and I146; modeling asurface bounded by the selected subset of amino acids; obtaining a testcompound 3D model of a test compound; matching the test compound 3Dmodel to the surface bounded by the selected subset of amino acids; andidentifying contact points between the selected subset of amino acids ofthe surface and the test compound 3D model. The method also optionallyfurther comprises determining a number of the contact points between thesurface and the test compound 3D model; and recording an affinity ratingfor the test compound 3D model corresponding to the number of contactpoints. In various embodiments, the method comprises determining a 3Drelationship between a selected subset of amino acids comprising F28,K29, A30, D32, I46, F47, and I55 (and, optionally A27, D31, K36, A37,I38, and/or F44), as well as R41, Y53, C59, E60, Q63, R65, E67, E71,K74, M75, N80, N82, R83, I84, I85, T87, F96, C106, C130, L131, N133,M134, N136, F137, E142, N145, and I146.

In another embodiment, the computer storage media has computerexecutable instructions that, when executed on the processor of acomputer, implement a method of comparing a peptide to selected threedimensional points (3D) in a TFPI Kunitz domain 1 protein (KD1), themethod comprising creating a protein structure for the KD1 protein;determining a three dimensional model of a selected subset of aminoacids in the KD1 protein, wherein the subset of amino acids comprisesPhe28, Lys29, Ala30, Asp32, Ile46, Phe47 and Ile55; determining a threedimensional model of a peptide; fitting the 3D model of the peptide tothe 3D model of the selected subset of amino acids; and generating anaffinity of the peptide for the selected subset of amino acids, whereinthe affinity is based on a number of amino acids in the subset incontact with the peptide and a bond strength at each contact point.Alternatively, the computer executable instructions implement a methodof comparing a peptide to selected three dimensional points (3D) in aTFPI Kunitz domain 1 and Kunitz domain 2 (KD1-KD2), the methodcomprising creating a protein structure for the KD1-KD2 protein;determining a three dimensional model of a selected subset of aminoacids in the KD1-KD2 protein, wherein the subset of amino acidscomprises R41, Y53, C59, E60, Q63, R65, E67, E71, K74, M75, N80, N82,R83, I84, I85, T87, F96, C106, C130, L131, N133, M134, N136, F137, E142,N145, and I146 (optionally in combination with F28, K29, A30, D32, I46,F47, and I55 (and, further optionally in combination with A27, D31, K36,A37, I38, and/or F44)); determining a three dimensional model of apeptide; fitting the 3D model of the peptide to the 3D model of theselected subset of amino acids; and generating an affinity of thepeptide for the selected subset of amino acids.

In addition, a method of comparing a test compound to selected threedimensional points in a TFPI KD1 protein or TFPI KD1-KD2 protein isprovided. The method comprises creating a protein structure for the KD1protein in a memory of a computer; determining a three dimensional modelof a selected subset of amino acids in the KD1 protein at a processor ofthe computer, wherein the selected subset of amino acids comprisesPhe28, Lys29, Ala30, Asp32, Ile46, Phe47, and Ile55; determining a threedimensional model of a test compound at the processor of the computer;fitting the 3D model of the test compound to the 3D model of theselected subset of amino acids at the processor of the computer; andgenerating an affinity of the test compound for the selected subset ofamino acids at the processor of the computer, wherein the affinity isbased on a number of amino acids in the subset in contact with the testcompound and a bond strength at each contact point. Alternatively, themethod comprises determining a three dimensional model of a selectedsubset of amino acids in the KD1-KD2 protein at a processor of thecomputer, wherein the selected subset of amino acids comprises R41, Y53,C59, E60, F96, C106, C130, N133, N136, F137, E142, Q63, R65, E67, E71,K74, M75, N80, N82, R83, I84, I85, T87, L131, M134, N145, and I146,optionally in combination with F28, K29, A30, D32, I46, F47, and I55(and, further optionally in combination with A27, D31, K36, A37, I38,and/or F44). The method further comprises, in some embodiments,displaying a 3D representation of the fit between the test compound andthe 3D model of the selected subset of amino acids and, optionally,repeating the steps described herein for a plurality of test compoundsand saving the respective affinities for each of the plurality of testcompounds.

With reference to FIG. 58, an exemplary system for implementing theclaimed method and apparatus includes a general purpose computing devicein the form of a computer 110. Components shown in dashed outline arenot technically part of the computer 110, but are used to illustrate theexemplary embodiment of FIG. 58. Components of computer 110 may include,but are not limited to, a processor 120, a system memory 130, amemory/graphics interface 121 and an I/O interface 122. The systemmemory 130 and a graphics processor 190 may be coupled to thememory/graphics interface 121. A monitor 191 or other graphic outputdevice may be coupled to the graphics processor 190.

A series of system busses may couple various system components includinga high speed system bus 123 between the processor 120, thememory/graphics interface 121 and the I/O interface 122, a front-sidebus 124 between the memory/graphics interface 121 and the system memory130, and an advanced graphics processing (AGP) bus 125 between thememory/graphics interface 121 and the graphics processor 190. The systembus 123 may be any of several types of bus structures including, by wayof example, and not limitation, such architectures include IndustryStandard Architecture (USA) bus, Micro Channel Architecture (MCA) busand Enhanced ISA (EISA) bus. As system architectures evolve, other busarchitectures and chip sets may be used but often generally follow thispattern. For example, companies such as Intel and AMD support the IntelHub Architecture (IHA) and the Hypertransport™ architecture,respectively.

The computer 110 typically includes a variety of computer readablemedia. Computer readable media can be any available media that can beaccessed by computer 110 and includes both volatile and nonvolatilemedia, removable and non-removable media. By way of example, and notlimitation, computer readable media may comprise computer storage media.Computer storage media includes both volatile and nonvolatile, removableand non-removable media implemented in any method or technology forstorage of information such as computer executable instructions, datastructures, program modules or other data. Computer storage mediaincludes RAM, ROM, EEPROM, flash memory or other memory technology,CD-ROM, digital versatile disks (DVD) or other optical disk storage,magnetic cassettes, magnetic tape, magnetic disk storage or othermagnetic storage devices or other physical storage elements thatphysically embody electronic data and excludes any propagated media suchas radio waves or modulated carrier signals.

The system memory 130 includes computer storage media in the form ofvolatile and/or nonvolatile memory such as read only memory (ROM) 131and random access memory (RAM) 132. The system ROM 131 may containpermanent system data 143, such as computer-specific configuration data.RAM 132 typically contains data and/or program modules that areimmediately accessible to and/or presently being operated on byprocessor 120. By way of example, and not limitation, FIG. 58illustrates operating system 134, application programs 135, otherprogram modules 136, and program data 137.

The I/O interface 122 may couple the system bus 123 with a number ofother busses 126, 127 and 128 that couple a variety of internal andexternal devices to the computer 110. A serial peripheral interface(SPI) bus 126 may connect to a basic input/output system (BIOS) memory133 containing the basic routines that help to transfer informationbetween elements within computer 110, such as during start-up.

A super input/output chip 160 may be used to connect to a number of‘legacy’ peripherals, such as floppy disk 152, keyboard/mouse 162, andprinter 196, as examples. The super I/O chip 160 may be connected to theI/O interface 122 with a bus 127, such as a low pin count (LPC) bus, insome embodiments. Various embodiments of the super I/O chip 160 arewidely available in the commercial marketplace. In one embodiment, bus128 may be a Peripheral Component Interconnect (PCI) bus.

The computer 110 may also include other removable/non-removable,volatile/nonvolatile computer storage media. By way of example only,FIG. 58 illustrates a hard disk drive 140 that reads from or writes tonon-removable, nonvolatile magnetic media. The hard disk drive 140 maybe a conventional hard disk drive.

Removable media, such as a universal serial bus (USB) memory 153,firewire (IEEE 1394), or CD/DVD drive 156 may be connected to the PCIbus 128 directly or through an interface 150. Otherremovable/non-removable, volatile/nonvolatile computer storage mediathat can be used in the exemplary operating environment include, but arenot limited to, magnetic tape cassettes, flash memory cards, digitalversatile disks, digital video tape, solid state RAM, solid state ROM,and the like.

The drives and their associated computer storage media discussed aboveand illustrated in FIG. 58, provide storage of computer readableinstructions, data structures, program modules and other data for thecomputer 110. In FIG. 58, for example, hard disk drive 140 isillustrated as storing operating system 144, application programs 145,other program modules 146, and program data 147. Note that thesecomponents can either be the same as or different from operating system134, application programs 135, other program modules 136, and programdata 137. Operating system 144, application programs 145, other programmodules 146, and program data 147 are given different numbers here toillustrate that, at a minimum, they are different copies. A user mayenter commands and information into the computer 20 through inputdevices such as a mouse/keyboard 162 or other input device combination.Other input devices (not shown) may include a microphone, joystick, gamepad, satellite dish, scanner, or the like. These and other input devicesare often connected to the processor 120 through one of the I/Ointerface busses, such as the SPI 126, the LPC 127, or the PCI 128, butother busses may be used. In some embodiments, other devices may becoupled to parallel ports, infrared interfaces, game ports, and the like(not depicted), via the super I/O chip 160.

The computer 110 may operate in a networked environment using logicalcommunication ports to one or more remote computers, such as a remotecomputer 180 via a network interface controller (NIC) 170. The remotecomputer 180 may be a personal computer, a server, a router, a networkPC, a peer device or other common network node, and typically includesmany or all of the elements described above relative to the computer110. The logical connection between the NIC 170 and the remote computer180 depicted in FIG. 58 may include a local area network (LAN), a widearea network (WAN), or both, but may also include other networks. Suchnetworking environments are commonplace in offices, enterprise-widecomputer networks, intranets, and the Internet.

FIG. 59 illustrates a 3D model of a TFPI protein 200 showingrepresentative amino acids 202, 204, 206 that comprise the TFPI protein.A specific region of the TFPI protein of interest is KD1, notspecifically illustrated. The surface shown is formed by the placementof the amino acids making up the protein. The surface of formed byspecific amino acids in the KD1 region are of interest when studying orcreating a TFPI inhibitor. As discussed in more detail herein, thebiological effects of KD1 are inhibited by binding certain amino acidsof within the KD1 region. Specifically, these target amino acids includeAla27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Ala37, Ile38, Phe44,Ile46, Phe47, and Ile55.

FIG. 60 illustrates a peptide 300 that binds to at least a portion ofthe target amino acids listed above.

FIG. 61 is an illustration of a method of performing KD1 and peptideinteraction modeling.

A 3D model of a protein may be obtained (block 302) and stored on amemory 140 of a computer 110. The model may be generated locally using aknown tool or may be obtained from a public source. In one embodimentthe protein is TFPI KD1 200.

A 3D relationship between a selected subset of amino acids in theprotein structure may be determined (block 304). In one embodiment, theselected subset of amino acids comprises Phe28, Lys29, Ala30, Asp32,Ile46, Phe47 and Ile55; and optionally further comprises Ala27, Asp31,Lys36, and Ile38; and optionally further comprises Ala37 and Phe44.Alternatively or in addition, the selected subset of amino acidscomprises R41, Y53, C59, E60, Q63, R65, E67, E71, K74, M75, N80, N82,R83, I84, I85, T87, F96, C106, C130, L131, N133, M134, N136, F137, E142,N145, and I146. Not every amino acid listed here is required for bindingto have an inhibitory (e.g., therapeutic) effect. That is, furthersubsets of this group may also have properties of interest.

For the particular subset of amino acids of interest, a surface boundedby the selected subset of amino acids may be modeled. An outer perimetermay be defined by those amino acids not having further amino acids ofinterest on each side. A texture of the surface may be defined by the 3Dlocation of each amino acid in the subset (block 306).

A 3D model of a candidate TFPI-binding compound (e.g., peptide) ofinterest may be generated and stored at a memory 140 of the computer 110(block 308).

The peptide 3D model may be matched or fitted to the surface bounded bythe selected subset of amino acids (block 310). A best fit between thetwo may be developed at the points of interest, that is, on the selectedamino acids of KD1. Several computer tools are available for such 3Dmodeling and fitting and may be used to create 3D models and match oneto another. One example is the HADDOCK tool described in: “de Vries, S.J., van Dijk, A. D. J., Krzeminski, M., van Dijk, M., Thureau, A., Hsu,V., Wassenaar, T. and Bonvin, A. M. J. J. (2007), HADDOCK versusHADDOCK: New features and performance of HADDOCK2.0 on the CAPRItargets. Proteins: Structure, Function, and Bioinformatics, 69: 726-733.doi: 10.1002/prot.21723”

The contact points between the model of the surface of the selectedsubset of amino acids of the surface and the test compound (e.g.,peptide) 3D model may be identified, stored, and optionally displayed ona monitor 191 of the computer 110 (block 312). A compound (e.g.,peptide) may be modified to increase the number of contact points or thestrength of the bonds at the contact points. To facilitate modeling thiseffect, a metric, described further below, may be developed to measurethe affinity of the compound to bind to the protein of interest, in ourexample, KD1.

Further, the contact points between the surface and the compound 3Dmodel may be counted (block 314) and an affinity rating for the compound3D model may be recorded corresponding to the number of contact points(block 316). For example, if all 14 of the above listed amino acids aretargeted and 12 of the 14 are actually contacted, or bound, by thecompound 3D model, an affinity rating of 12/14 or 0.86 may be calculatedand recorded.

However, the affinity rating as a measure of how tightly a candidatecompound is coupled, and therefore, how long it may stay coupled to KD1may be more accurately described in terms of not only the number ofbonds of interest but also the type of bond. The bond type for eachcontact point may also be determined (block 318). With respect toTFPI-binding peptides, hydrophobic bonds having an intermoleculardistant of ≦4 angstroms may be differentiated from bonds with anintermolecular distance of 2.6-3.2 angstroms. In one embodiment, bondsless than 3.2 angstroms may be assigned a weight of 1.5 and bonds >than3.2 angstroms may be assigned a weight of 1.25. The affinity rating maybe updated or recalculated in view of the bond type using this, oranother weighting (block 320). For example, if, in the previous example,5 of the bonds are short bonds and 7 of the bonds are long bonds, thenew affinity rating may be (5*1.5+7*1.25)/14=1.16.

If only 7 amino acids from KD1 are targeted and 4 connect with shortbonds, the affinity rating may be (4*1.5)/7=0.86. However, in this casethe fewer targeted amino acids will be considered when comparisons aremade to other affinity ratings. For example, all ratings could benormalized to a standard based on total desired target sites.

If no more iterations are to be performed the no branch from block 322may be taken and the results of may be stored for future analysis anddecision making (block 324). If additional peptides, or variants of thepreviously tested peptide, are to be analyzed, the yes branch from block322 may be taken and a new or updated model of the peptide of interestmay be generated or otherwise obtained and stored (block 326). The stepsat blocks 310 to 320 may be repeated and the results of the current runmay be compared to results from previous runs to determine whichpeptides/variants have higher affinity ratings and merit more work,including possible physical testing.

The ability to target particular sites with 3D modeling and to generatea comparative rating allows hundreds, if not thousands of samples to beprocessed and compared with relative ease, avoiding the time and cost ofx-ray crystallography. This technique may be particularly applicable tomodeling associated with the F28, K29, A30, D32, I46, F47, I55, A27,D31, K36, I38, F2, A37 and F44 amino acids from TFPI KD1 and/or R41,Y53, C59, E60, Q63, R65, E67, E71, K74, M75, N80, N82, R83, I84, I85,T87, F96, C106, C130, L131, N133, M134, N136, F137, E142, N145, and I146from TFPI KD1-KD2.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. In addition, the entiredocument is intended to be related as a unified disclosure, and itshould be understood that all combinations of features described hereinare contemplated, even if the combination of features are not foundtogether in the same sentence, or paragraph, or section of thisdocument. For example, where protein therapy is described, embodimentsinvolving polynucleotide therapy (using polynucleotides/vectors thatencode the protein) are specifically contemplated, and the reverse alsois true. Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. The invention includes, for instance,all embodiments of the invention narrower in scope in any way than thevariations specifically mentioned above. With respect to aspects of theinvention described as a genus, all individual species are individuallyconsidered separate aspects of the invention. With respect to aspects ofthe invention described or claimed with “a” or “an,” it should beunderstood that these terms mean “one or more” unless contextunambiguously requires a more restricted meaning. With respect toelements described as one or more within a set, it should be understoodthat all combinations within the set are contemplated. Finally, unless aclaim element is defined by reciting the word “means” and a functionwithout the recital of any structure, it is not intended that the scopeof any claim element be interpreted based on the application of 35U.S.C. §112, sixth paragraph.

EXAMPLES

The invention, thus generally described, will be understood more readilyby reference to the following examples, which are provided by way ofillustration and are not intended to limit the invention.

Example 1

The following example describes production, identification, andscreening of peptides for binding to TFPI.

Peptides candidates were obtained from commercial suppliers (e.g.,PolyPeptide Laboratories SAS (Strasbourg, France) and JPT PeptideTechnologies GmbH (Berlin, Germany)). Methods for synthesizing candidatepeptides are provided above. Candidate peptides were synthesized astrifluoroacetate (TFA) salts with a purity >90% or >60%. All peptideswere solved in DMSO to a stock concentration of 10 mM. TFPI-bindingpeptide sequences were identified using an mRNA display library. ThemRNA display technology is superior to other library screeningtechniques for allowing for a diversity of 10¹⁴ different sequenceswithin a starting pool and avoiding, e.g., the in vivo steps requiredfor phage display. In brief, the technology involves directly linkingmRNA to its encoded candidate peptide through a puromycin molecule (FIG.5). The mRNA display method is further described in International PatentPublication No. WO 2005/051985 and Liu et al., Methods in Enzymology,318, 268-293 (2000). TFPI was immobilized to a solid support via biotinand exposed to candidate peptide-RNA complexes. TFPI-bound candidatepeptide-RNA complexes were isolated, and the RNA reverse transcribed toobtain coding DNA. High affinity binders were obtained following six toten selection rounds using a competitive elusion strategy. Many of thecandidate peptides were 31 amino acids in length (27 randomized aminoacids and 2 amino acids flanking both termini).

Selected peptides were synthesized and subjected to peptide optimizationusing a microarray-based scan analysis to identify peptide fragmentsretaining TFPI-binding affinity. For example, a microarray-based scan ofJBT0047 was performed using a series of 20 amino acid fragments of thepeptide, the sequences of which overlapped by 19 amino acids. Briefly,N-terminally, aminooxyacetate-modified peptides were printed on Corningepoxide glass slides. After washing and drying, the slides were treatedin a TECAN HS400™ incubation station. Slides were washed for two minutesin Tris-buffered saline with 0.1% TWEEN 20® (TBST), and blocked for 30minutes in Tris-based, T-20 SuperBlock™ buffer (5 mM CaCl₂) (Pierce).After blocking, the slides were washed for 2.5 minutes in TBST. Theslides were subsequently incubated with DYLIGHT™ 649-labeled TFPI (1μg/ml in Tris-based, T-20 SuperBlock™ buffer (5 mM CaCl₂)) for 45minutes, and washed twice with continuous flow TBST for ten minutes. Theslides were subjected to a final wash with saline-sodium citrate bufferfor two minutes, and air-dried for four minutes. The slides were scannedin an Axon GenePix® 4000B scanner, and scans were analyzed using theGenePix® Pro software. N- and C-terminal truncation analysissupplemented the scan analysis. The microarray scan results demonstratedthat peptide JBT0293 bound TFPI with the highest affinity. A series ofsubstitution mutants based on the amino acid sequence of JBT0293 wasgenerated and tested for TFPI binding properties.

The affinity of a subset of peptides for TFPI was demonstrated via anenzyme-linked immunosorbent assay (ELISA)-like assay (binding (EC₅₀)ELISA) performed with biotinylated peptides. Ninety-six well MaxiSorpplates (Nunc) were coated with 3 μg/mL TFPI in coating buffer (15 mMNa₂CO₃, 35 mM NaHCO₃, pH 9.6) over night. Plates were washed three timeswith 350 μl wash buffer (HNaT: 175 mM NaCl, 25 mM HEPES, 5 mM CaCl₂,0.1% Tween 80, pH 7.35), and subsequently blocked with 200 μl 2% yeastextract in HNaT for 2 hours. Plates were then washed three times with350 μl HNaT. Biotinylated candidate peptides were diluted from a DMSOstock 1/200 in HNaT. The initial peptide concentration was 50 μM if noprecipitate appeared during the 1/200 dilution of the 10 mM peptidestock solution. Pre-dilutions of the peptide stock in DMSO wereconducted if precipitates formed. The diluted peptides were applied tothe Maxisorp plates, serial dilutions (1/3) were generated, and thedilutions were incubated for 1.5 hours at room temperature. Incubationwas followed by three wash steps (350 μl HNaT). Bound peptide wasdetected by incubation with horseradish peroxidase-conjugatedstreptavidin (1 hour), followed by three wash steps with HNaT and asubsequent chromogenic conversion of added TMB(3,3′5,5′-Tetramethylbenzidin). The assay is illustrated in FIG. 6A.

Generally, peptide binding to immobilized TFPI was significantly abovebackground. EC₅₀ values for biotinylated peptides are given in FIGS.32-39. The binding curve of one TFPI-binding peptide, JBT0132, isdepicted in FIG. 7. The EC₅₀ of JBT0132 was calculated to be about 2.2nM.

In addition, a competition (IC₅₀) ELISA was performed using biotinylatedTFPI-binding peptides as “tracers” to compete for TFPI-binding withnon-biotinylated candidate peptides. The assay principle is depicted inFIG. 6B. Ninety-six well MaxiSorp plates (Nunc) were coated with 3 μg/mLTFPI in coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) over night.The concentration of TFPI can be altered depending on the particularconditions of the assay; in other IC₅₀ ELISA assays referenced herein,the coating buffer contained 0.05 μg/ml TFPI. Plates were washed threetimes with 350 μl wash buffer (HNaT: 175 mM NaCl, 25 mM HEPES, 5 mMCaCl₂, 0.1% Tween 80, pH 7.35), and blocked with 200 μl 2% yeast extractin HNaT for 2 hours. Plates were then washed three times with 350 μlHNaT. Biotinylated tracer peptides were applied at a concentrationcorresponding to their respective EC₉₀ values determined in the bindingELISA (median if n>2). A competitor stock solution of peptide (10 mM)was diluted 1/33.3 in HNaT without HSA, and a serial 1/3 dilution wasprepared with HNaT with 3% DMSO. The dilution strategy employed in aparticular assay will depend on the affinity of the peptides. Thedilution was further diluted with the biotinylated tracer peptide in aratio of 1:6 (20 μl competitor dilution and 100 μl tracer peptide). Themixture of competitor and tracer peptide was applied to the TFPI-coatedmicrotiter plate and incubated for 1.5 hours. The plates were washedthree times with 350 μl HNaT. Peptide-TFPI binding was detected byapplying HRP-conjugated streptavidin to the microtiter plate, incubatingthe mixture for one hour, washing the plate three times with 350 μlHNaT, applying TMB (3,3′5,5′-Tetramethylbenzidin), and detecting thesubsequent chromogenic conversion of TMB by HRP. IC₅₀ graphs forrepresentative non-biotinylated peptides are provided in FIGS. 8A-8D.IC₅₀ measurements of peptides JBT0303, JBT0120, and JBT0224 are setforth in Table 3.

TABLE 3 Tracer Tracer Peptide IC₅₀ [μM] n SD Peptide Concentration [μM]JBT0303 0.119 2 0.064 JBT0131 0.0409 JBT0120 0.0189 3 0.0044 JBT01240.0718 JBT0224 n.a. 1 JBT0126 0.240

In addition to the competition ELISA (IC₅₀) assay, a screening assay wasemployed to measure higher numbers of peptides in parallel. Thescreening ELISA is similar to the competition IC₅₀ ELISA with theexception that only three different concentrations of the competitorwere employed (300 nM, 100 nM and 33.3 nM for the JBT0047 class, and50000 nM, 16667 nM and 5556 nM for the JBT0122 class). In someinstances, screening results were expressed as percent inhibition of thetracer signal in relation to a competitive peptide (competitive peptideJBT0477 for the JBT0047 family, and competitive peptide JBT1697 for theJBT0122 family). The competition IC₅₀ assay results and the screeningassay results of peptides prepared and screened in accordance with themethods set forth herein are provided in FIGS. 32-39. The mean IC₅₀values presented in FIGS. 32-39 are based on a greater number of assaysthan the values presented in Table 3 and, therefore, the values maydiffer slightly. The results of the screening ELISA are presented aspercent inhibition of tracer peptide JBT0131 binding. Several peptidesthat were analyzed using the IC₅₀ ELISA are classified in FIGS. 32-39according to their binding affinity as set forth in Table 4.

TABLE 4 TFPI competition ELISA IC₅₀ [nM] Group <50 nM A  50 ≦ x <100 nMB 100 ≦ x <250 nM C  250 ≦ x <1000 nM D 1000 ≦ x <5000 nM E  5000 ≦ x<10000 nM F 10000 ≦ x <50000 nM G

Exemplary TFPI-binding peptides identified using the methods describedherein are presented in Table 5. Some peptides were biotinylated, andmany comprise N- and C-terminal lysines to promote solubility. Severalpeptides exhibited TFPI-inhibitory activity in model and/or plasmaticassay systems, as described below.

TABLE 5 Peptide Parent Sequence SEQ ID JBT0047 QSKKNVFVFGYFERLRAK 1JBT0047 JBT0047 Ac-SGVGRLQVAFQSKKNVFVFGYFERLRAKLTS-NH2 253 JBT0051JBT0047 Biotinyl-Ttds-SGVGRLQVAFQSKKNVFVFGYFERLRAKLTS-NH2 962 JBT0055JBT0047 Ac-SGVGRLQVAFQSKKNVFVFGYFERLRAKLTS-Ttds-Lys(Biotinyl)- 963 NH2JBT0131 JBT0047 Biotinyl-Ttds-AFQSKKNVFVFGYFERLRAK-NH2 964 JBT0132JBT0047 Biotinyl-Ttds-FQSKKNVFVFGYFERLRAKL-NH2 965 JBT0133 JBT0047Biotinyl-Ttds-QSKKNVFVFGYFERLRAKLT-NH2 966 JBT0155 JBT0047Ac-KKSGVGRLQVAFQSKKNVFVFGYFERLRAKLTSKK-NH2 8 JBT0158 JBT0047Ac-KKSGVGRLQVAFQSKKNVFVFGYFERLRAKKK-NH2 9 JBT0162 JBT0047Ac-KKGRLQVAFQSKKNVFVFGYFERLRAKLTSKK-NH2 10 JBT0163 JBT0047Ac-KKQVAFQSKKNVFVFGYFERLRAKLTSKK-NH2 11 JBT0164 JBT0047Ac-KKFQSKKNVFVFGYFERLRAKLTSKK-NH2 12 JBT0166 JBT0047Biotinyl-Ttds-KKFQSKKNVFVFGYFERLRAKLKK-NH2 968 JBT0169 JBT0047Ac-KKAFQSKKNVFVFGYFERLRAKKK-NH2 254 JBT0170 JBT0047Ac-KKFQSKKNVFVFGYFERLRAKLKK-NH2 13 JBT0171 JBT0047Ac-KKQSKKNVFVFGYFERLRAKLTKK-NH2 255 JBT0174 JBT0047Ac-KKAFQSKKNVFVFGYFERLRAKLKK-NH2 14 JBT0175 JBT0047Ac-KKAFQSKKNVFVFGYFERLRAKLTKK-NH2 182 JBT0293 JBT0047Ac-FQSKKNVFVFGYFERLRAKL-NH2 256 X₃X₄X₅KX₇NVFX₁₁X₁₂GYX₁₅X₁₆RLRAKX₂₂ 2JBT0294 JBT0047 Ac-YQSKKNVFVFGYFERLRAKL-NH2 257 JBT0295 JBT0047Ac-FSSKKNVFVFGYFERLRAKL-NH2 713 JBT0296 JBT0047Ac-FQNKKNVFVFGYFERLRAKL-NH2 407 JBT0297 JBT0047Ac-FQSKNNVFVFGYFERLRAKL-NH2 183 JBT0298 JBT0047Ac-FQSKQNVFVFGYFERLRAKL-NH2 747 JBT0299 JBT0047Ac-FQSKKNVFAFGYFERLRAKL-NH2 408 JBT0300 JBT0047Ac-FQSKKNVFSFGYFERLRAKL-NH2 409 JBT0301 JBT0047Ac-FQSKKNVFTFGYFERLRAKL-NH2 470 JBT0302 JBT0047Ac-FQSKKNVFVAGYFERLRAKL-NH2 258 JBT0303 JBT0047Ac-FQSKKNVFVDGYFERLRAKL-NH2 184 JBT0304 JBT0047Ac-FQSKKNVFVLGYFERLRAKL-NH2 259 JBT0305 JBT0047Ac-FQSKKNVFVQGYELRLRAKL-NH2 260 JBT0306 JBT0047Ac-FQSKKNVFVSGYFERLRAKL-NH2 185 JBT0307 JBT0047Ac-FQSKKNVFVYGYFERLRAKL-NH2 261 JBT0308 JBT0047Ac-FQSKKNVFVFGYKERLRAKL-NH2 411 JBT0309 JBT0047Ac-FQSKKNVFVFGYYERLRAKL-NH2 412 JBT0310 JBT0047Ac-FQSKKNVFVFGYFDRLRAKL-NH2 262 JBT0311 JBT0047Ac-FQSKKNVFVFGYFERLRAKN-NH2 748 TFVDERLLYFLTIGNMGMYAAQLKF 3 JBT0049JBT0049 Ac-SGNTFVDERLLYFLTIGNMGMYAAQLKFRTS-NH2 3025 JBT0053 JBT0049Biotinyl-Ttds-SGNTFVDERLLYFLTIGNMGMYAAQLKFRTS-NH2 3006 JBT0057 JBT0049Ac-SGNTFVDERLLYFLTIGNMGMYAAQLKFRTS-Ttds-Lysin(biotin)- 3018 NH2 JBT0190JBT0049 Ac-KKSGNTFVDERLLYFLTIGNMGMYAAQLKFRTSKK-NH2 3031 JBT0193 JBT0049Ac-KKSGNTFVDERLLYFLTIGNMGMYAAQLKFKK-NH2 3073 JBT0197 JBT0049Ac-KKTFVDERLLYFLTIGNMGMYAAQLKFRTSKK-NH2 3076 VIVFTFRHNKLIGYERRY 4JBT0050 JBT0050 Ac-SGRGCTKVIVFTFRHNKLIGYERRYNCTS-NH2 3047 JBT0054JBT0050 Biotinyl-Ttds-SGRGCTKVIVFTFRHNKLIGYERRYNCTS-NH2 3002 JBT0058JBT0050 Ac-SGRGCTKVIVFTFRHNKLIGYERRYNCTS-Ttds-Lysin(biotin)-NH2 3003JBT0129 JBT0050 Ac-SGRG[CTKVIVFTFRHNKLIGYERRYNC]TS-NH2 3026 JBT0130JBT0050 Biotinyl-Ttds-SGRG[CTKVIVFTFRHNKLIGYERRYNC]TS-NH2 3001 JBT0205JBT0050 Ac-KKSGRGCTKVIVFTFRHNKLIGYERRYNCTSKK-NH2 3029 JBT0208 JBT0050Ac-KKSGRGCTKVIVFTFRHNKLIGYERRYNKK-NH2 3027 JBT0211 JBT0050Ac-KKGCTKVIVFTFRHNKLIGYERRYNCTSKK-NH2 3032 JBT0212 JBT0050Ac-KKKVIVFTFRHNKLIGYERRYNCTSKK-NH2 3033 JBT0217 JBT0050Ac-KKTKVIVFTFRHNKLIGYERRYKK-NH2 3062 JBT0218 JBT0050Ac-KKKVIVFTFRHNKLIGYERRYNKK-NH2 3063 JBT0219 JBT0050Ac-KKVIVFTFRHNKLIGYERRYNCKK-NH2 3030 GVWQTHPRYFWTMWPDIKGEVIVLFGT 5JBT0101 JBT0101 Ac-KKSGVWQTHPRYFWTMWPDIKGEVIVLFGTSKK-NH2 3036 JBT0052JBT0101 Biotinyl-Ttds-KKSGVWQTHPRYFWTMWPDIKGEVIVLFGTSKK-NH2 3004 JBT0103JBT0101 Ac-KKSGVWQTHPRYFWTMWPDIKGEVIVLFGTS-Ttds-KK- 3005Lysin(biotinyl)-NH2 JBT0178 JBT0101Ac-KKSGVWQTHPRYFWTMWPDIKGEVIVLFGTKK-NH2 3028 JBT0182 JBT0101Ac-KKGVWQTHPRYFWTMWPDIKGEVIVLFGTSKK-NH2 3037 KWFCGMRDMKGTMSCVWVKF 6JBT0120 JBT0120 Ac-SGASRYKWF[CGMRDMKGTMSC]VWVKFRYDTS-NH2 1047 JBT0124Biotinyl-Ttds-SGASRYKWF[CGMRDMKGTMSC]VWVKFRYDTS-NH2 1290 JBT0247 JBT0120Ac-SGASRYKWFCGMRDMKGTMSCVWVKFRYDTS-NH2 1213 JBT0248 JBT0120Ac-KKSGASRYKWF[CGMRDMKGTMSC]VWVKFRYDTSKK-NH2 1001 JBT0251 JBT0120Ac-KKKWFCGMRDMKGTMSCVWVKFKK-NH2 1202 JBT0252 JBT0120Ac-KKCGMRDMKGTMSCVWVKFRYDKK-NH2 1215 ASFPLAVQLHVSKRSKEMA 7 JBT0122JBT0122 Ac-SGYASFPLAVQLHVSKRSKEMALARLYYKTS-NH2 2002 JBT0126 JBT0122Biotinyl-Ttds-SGYASFPLAVQLHVSKRSKEMALARLYYKTS-NH2 2498 JBT0221 JBT0122Ac-KKSGYASFPLAVQLHVSKRSKEMALARLYYKTSKK-NH2 2003 JBT0224 JBT0122Ac-KKSGYASFPLAVQLHVSKRSKEMALARLYYKK-NH2 2298 JBT0225 JBT0122Ac-KKSGYASFPLAVQLHVSKRSKEMALARKK-NH2 2128 JBT0226 JBT0122Ac-KKSGYASFPLAVQLHVSKRSKEMAKK-NH2 2299 JBT0228 JBT0122Ac-KKASFPLAVQLHVSKRSKEMALARLYYKTSKK-NH2 2016 JBT0232 JBT0122Ac-KKGYASFPLAVQLHVSKRSKEMKK-NH2 2303 JBT0233 JBT0122Ac-KKYASFPLAVQLHVSKRSKEMAKK-NH2 2304

This example provides exemplary methods of generating and characterizingTFPI-binding peptides (e.g., TFPI-inhibitory peptides). All peptides inTable 5 were found to bind human TFPI-1α. Mutation analysis demonstratedthat at least one amino acid in a TFPI-binding peptide may besubstituted while retaining affinity for TFPI. The peptides of Table 5tested in ELISA assays bound TFPI-1α with an EC₅₀ of less than 10 μM(1×10⁻⁵ M) and an IC₅₀ of less than 50 μM.

Example 2

Selected TFPI-binding peptides were further characterized in terms of“anti-target” binding. This example demonstrates that TFPI-bindingpeptides exhibit reduced affinity for non-TFPI-1 proteins.

TFPI-2 was selected as an anti-target because of its similarity toTFPI-1. The binding kinetics of TFPI-binding peptides to human TFPI-1(residues 29-282 fused at the C-terminus to a 10 His-tag; MW 41 kDa (R&DSystems, Minneapolis, Minn.; catalog number 2974-PI)) murine TFPI-1(residues 29-289 fused at the C-terminus to a 10 His-tag; MW 41 kDa (R&DSystems; catalogue number 2975-PI)), and TFPI-2 (R&D Systems,Minneapolis, Minn.) were studied using a BIAcore 3000™ surface plasmonresonance assay (GE Healthcare, Chalfont St. Giles, UK). TFPI proteinswere immobilized on a C1 chip (GE Healthcare, Order Code: BR-1005-40) byamine coupling chemistry aiming for 500 RU. Several TFPI-bindingpeptides were employed as analytes for interacting with the immobilizedTFPI proteins. A flow rate of 30 μl/min was utilized. After 180 seconds,180 μl of peptide solution was injected at six different concentrationsranging from 3.84 nM to 656.25 nM, followed by a dissociation time of480 seconds. The chip was regenerated with 45 μl 10 mM NaOH. Eachbinding experiment was preceded and followed by four measurements withHBS-P buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% P20) plus 1% DMSOand 0.8% P80. BIAevaluation® Version 4.1 software (GE Healthcare) wasemployed to analyze the data. Sensorgrams were fitted to a 1:1 Langmuirbinding curve to determine k_(on) and k_(off) and calculate K_(D).

Certain tested peptides, e.g., JBT0050, JBT0121, JBT0205 and JBT0211,bound to the blank cell and binding constants from those sensorgramscould not be determined. JBT0133 showed weak binding to TFPI-1.Sensorgrams from other peptides gave reliable binding constants. Resultsfrom BIAcore analysis of several TFPI-binding peptides is provided inTable 6 and FIGS. 19-21. Each of the peptides listed in Table 6presented a K_(D) of less than 10 μM. In addition to the peptides listedbelow, JBT0375 and JBT0477, substitution mutants of JBT0293 at aminoacid position 5 (JBT0375) or amino acid positions 5 and 10 (JBT0477),also exhibited a K_(D) of less than 10 μM. JBT1837 (SEQ ID NO: 1044)also demonstrated a K_(D) of less than 10 μM (K_(D)=0.5 nM; k_(on)=8×10³1/Ms; k_(off)=1.3×10⁻⁶ 1/s). Sensorgrams of two of the peptides areprovided as FIGS. 9A and 9B.

TABLE 6 Peptide k_(on) (1/Ms) k_(off) (1/s) K_(D) (M) JBT0047 4.0 × 10⁵1.9 × 10⁻² 4.7 × 10⁻⁸ JBT0120 1.17 × 10⁶  4.78 × 10⁻²  4.08 × 10⁻⁸ JBT0131 1.4 × 10⁵ 6.0 × 10⁻² 4.31 × 10⁻⁷  JBT0132 3.55 × 10⁴  3.26 ×10⁻²  9.17 × 10⁻⁷  JBT0224 6.39 × 10⁴  1.95 × 10⁻²  3.05 × 10⁻⁷  JBT02936.0 × 10⁵ 5.6 × 10⁻² 9.5 × 10⁻⁸ JBT0297 5.0 × 10⁵ 1.4 × 10⁻² 2.9 × 10⁻⁸JBT0303 8.13 × 10⁵  2.75 × 10⁻²  3.4 × 10⁻⁸ JBT0305 7.5 × 10⁵ 3.1 × 10⁻²6.1 × 10⁻⁸

Interaction with the TFPI-2 anti-target also was examined. The maximumsignal generated from candidate peptide interaction with human TFPI-2was much lower than the signals obtained with TFPI-1 as an interactionpartner. Kinetic analysis of the low TFPI-2 binding signals was prone toerror; therefore, visual comparison of sensorgrams was used to estimatebinding affinity. A sensorgram illustrating JBT0120 binding to TFPI-1and TFPI-2 is provided as FIGS. 10A and 10B. JBT0120 binds TFPI-2 with10-fold lower affinity compared to its binding affinity for TFPI-1.JBT0132 also was found to exhibit at least 10-fold greater affinity forTFPI-1 than TFPI-2.

The data provided by this example confirm that TFPI-binding peptidesspecifically bind TFPI-1.

Example 3

The following example describes the characterization of TFPI-inhibitoryactivity of select peptides identified in Example 1 using FXa inhibitionand extrinsic tenase inhibition assays. Both assays are predictive ofactivity in plasmatic systems. The extrinsic tenase assay gives insightinto the influence of the peptides on (a) the interaction of FXa andTFPI and (b) the interaction of the FXa-TFPI complex with the TF-FVIIacomplex. The FXa inhibition assay measures a peptide's influence on theinteraction of FXa and TFPI only.

The extrinsic tenase complex is responsible for FX and FIX activationupon initiation of the coagulation process. The extrinsic complex iscomposed of FVIIa, Tissue Factor (TF), and FX substrate. To determinethe influence of peptides on the TFPI-mediated inhibition of theextrinsic tenase complex, a coupled enzyme assay was established.Peptides were diluted 1/6.25 from 10 mM stocks (in DMSO) and furtherdiluted by serial 1/4 dilutions in buffer or DMSO to prevent unwantedprecipitation. TFPI was diluted in HNaCa-HSA or BSA (25 mM HEPES; 175 mMNaCl; 5 mM CaCl₂; 0.1% HSA or BSA; pH 7.35). FVIIa, lipidated TF,phospholipid vesicles (DOPC/POPS 80/20), and chromogenic substratespecific for FXa (S-2222 (available from DiaPharma, West Chester,Ohio)), all diluted in HNaCa-HSA, were added to 96-well plates. After anincubation period, TFPI and peptide dilutions were added, resulting in afinal concentration of 2.5% DMSO (if present in the peptide stock). FXactivation was initiated by adding FX to the wells. FXa-mediatedchromogenic substrate conversion was determined by observing an increasein absorbance using a micro-plate reader. The amount of FXa generated atcertain time points was calculated from the OD readings. FXa generatedat 20 minutes after start of the reaction was considered for calculationof EC₅₀ from plots of peptide concentration versus the inhibition ofTFPI (%).

The functional inhibition of TFPI also was examined using a FXainhibition assay. A FXa-specific chromogenic substrate (S-2222) andTFPI, both diluted in HNaCa-HSA, were added to 96 well plates. Peptideswere diluted 1/6.25 from 10 mM stocks (in DMSO or Aqua-Dest) and furtherdiluted by serial 1/4 dilutions in buffer or DMSO to prevent unwantedprecipitation. The peptide dilutions (2.5 μl) were added to the 96 wellplates, resulting in a final concentration of 2.5% DMSO (if present inthe peptide stock). The conversion of chromogenic substrate wastriggered by the addition of FXa, and the kinetics of the conversionwere measured in a micro-plate reader. Because TFPI inhibits FXa slowly,OD readings after 115 minutes were considered for calculation of theEC₅₀ from plots of peptide concentration versus the inhibition of TFPI(%).

Results from the extrinsic tenase assay and FXa inhibition assay areprovided in Table 7 and FIGS. 22-27.

TABLE 7 FXa Inhibition Assay Extrinsic Tenase Assay % inhibition %inhibition EC₅₀ [μM] @ 2.5 μM EC₅₀ [μM] @ 2.5 μM JBT0120 0.9 45 0.9 45JBT0132 1.2 36 0.1 10 JBT0224 n.a. 26 3.5 18 JBT0303 1.2 61 n.a. 8

Referring to Table 7, JBT0120, JBT0132, and JBT0224 restored extrinsiccomplex-mediated FX activation in the presence of TFPI-1 with an EC₅₀ of<2 μM, resulting in between about 20% to about 60% inhibition of TFPIactivity. JBT0047 (EC₅₀=1.4 μM), JBT0131 (EC₅₀=2.2 μM), and JBT0293(EC₅₀=2.9 μM) also restored extrinsic complex activity in the presenceof TFPI-1. In addition, JBT0120, JBT0132, JBT0224, and JBT0303 restoredFXa activity in the presence of TFPI-1 with an EC₅₀ of <5 μM, resultingin between about 5% to about 50% inhibition of TFPI activity, in the FXainhibition assay. JBT0047 (EC₅₀=0.7 μM), JBT0131 (EC₅₀=8.2 μM), JBT0293(EC₅₀=1.3 μM), JBT0297 (EC₅₀=0.6 μM), and JBT0305 (EC₅₀=2.3 μM) alsorestored activity of FXa in the presence of TFPI-1 in the FXa inhibitionassay. This example confirms that peptides of the invention are TFPIantagonists.

Example 4

In this example, the TFPI inhibitory activity of peptides is establishedusing a plasma-based assay.

The influence of peptides on thrombin generation was measured induplicate via calibrated automated thrombography in a Fluoroskan Ascent®reader (Thermo Labsystems, Helsinki, Finland; filters 390 nm excitationand 460 nm emission) following the slow cleavage of thethrombin-specific fluorogenic substrate Z-Gly-Gly-Arg-AMC (Hemker,Pathophysiol. Haemost. Thromb., 33, 4-15 (2003)). Plasma from patientswith FVIII or FIX deficiency (George King Bio-Medical Inc., OverlandPark, KN) was obtained for testing. The residual coagulation factoractivity for each of the plasmas was lower than 1%. As a model forantibody-mediated FVIII deficiency, frozen pooled normal plasma (GeorgeKing Bio-Medical Inc., Overland Park, KN) was incubated with high titer,heat inactivated, anti-human FVIII plasma raised in goat (4490 BU/ml;Baxter BioScience, Vienna, Austria) giving rise to 50 BU/mL. The plasmaswere mixed with corn trypsin inhibitor (CTI) (Hematologic Technologies,Inc., Essex Junction, Vt.) to inhibit Factor XIIa contamination,resulting in a final concentration of 40 μg/mL.

Pre-warmed (37° C.) plasma (80 μL) was added to each well of a 96 wellmicro-plate (Immulon 2HB, clear U-bottom; Thermo Electron, Waltham,Mass.). To trigger thrombin generation by Tissue Factor, 10 μL of PPPlow reagent containing low amounts (12 pM) of recombinant human TissueFactor and phospholipid vesicles composed of phosphatidylserine,phosphatidylcholine and phosphatidylethanolamine (48 μM) (ThrombinoscopeBV, Maastricht, The Netherlands) were added. Peptides were diluted 1/7.5from 10 mM stocks with DMSO, and further diluted 1/8.33 with Aqua-Destresulting in a DMSO concentration of 12%, providing a 0.5% DMSOconcentration in the final assay mix. Just prior putting the plate intothe pre-warmed (37° C.) reader, 5 μL of HEPES buffered saline with 5mg/mL human serum albumin (Sigma-Aldrich Corporation, St. Louis, Mo.,USA) or 12% DMSO in Aqua-Dest was added, followed by addition of thepeptide dilutions or reference proteins (FVIII Immunate referencestandard (Baxter BioScience, Vienna, Austria); Factor VIII InhibitorBy-Passing Activity (FEIBA) reference standard (Baxter BioScience,Vienna, Austria); NovoSeven (Novo Nordisk, Denmark); and purified humanplasma FIX (Enzyme Research Laboratories, South Bend, Ill.)). Thrombingeneration was initiated by dispensing into each well 20 μL of FluCareagent (Thrombinoscope BV, Maastricht, The Netherlands) containing afluorogenic substrate and HEPES-buffered CaCl₂ (100 mM). Fluorescenceintensity was recorded at 37° C.

The parameters of the resulting thrombin generation curves werecalculated using Thrombinoscope™ software (Thrombinoscope BV,Maastricht, The Netherlands) and thrombin calibrator to correct forinner filter and substrate consumption effects (Hemker, Pathophysiol.Haemost. Thromb., 33, 4-15 (2003)). For calculating the thrombingenerating activity of certain peptide concentrations equivalent to thereference proteins (e.g., FVIII Immunate® reference standard, FEIBAreference standard), the thrombin amounts at the peak of each thrombingeneration curve (peak thrombin, nM) were plotted against the standardconcentrations, and fitted by a non-linear algorithm. Based on thiscalibration, FVIII Immunate, FIX, FEIBA or NovoSeven equivalentactivities were calculated. Results for various peptides are provided inFIGS. 12-18 and 28-30. Representative results are provided in Table 8.(* denotes that FVIII deficient plasma was obtained from a differentdonor.)

TABLE 8 % FVIII-equivalent activity in FEIBA-equivalent activity inFVIII deficient plasma @ FVIII inhibited plasma @ 10 μM peptide 10 μMpeptide [mU/ml] JBT0120 37.4* 298 JBT0132 5.3 41 JBT0224 16.2 191JBT0303 20.8 253

With reference to Table 8, JBT0120, JBT0132, JBT0224, and JBT0303improved TFPI-dependent thrombin generation in FVIII-depleted plasma tolevels exceeding 1% of the level of thrombin generation in plasmacontaining FVIII (% FVIII-equivalent activity). The tested peptidesexhibited approximately 5%-40% FVIII-equivalent activity inFVIII-deficient plasma. JBT0120 and JBT0132 improved peak thrombin andpeak time, dose dependently, as illustrated in FIGS. 11A and 11B.

Substitution mutants based on the amino acid sequence of JBT0293 alsowere tested in a plasma-based assay, as well as the FXa inhibition andextrinsic tenase inhibition assay described in Example 3. Representativeresults are provided in Table 9.

TABLE 9 FVIII- equivalent Extrinsic activity FXa Tenase (mU/ml) inBiacore Inhibition Inhibition Hem A K_(D) EC₅₀ EC₅₀ plasma @ 1 (nM) (μM)(μM) μM peptide JBT0047 47 0.7 1.4 45 JBT0293 97 1.3 2.9 48 JBT0303 341.2 NA 125 JBT0500 8.2 0.12 — 372 JBT0740 2.4 0.07 — 333 JBT1584 0.30.01 — 489

Additionally, JBT0477, which comprises the amino acid sequence ofJBT0293 but for substitutions at amino acid positions 5 and 10 of theJBT0293 sequence, improves thrombin generation equivalent to 413 mU/mlof FVIII (at 1 μM of peptide) in FVIII-deficient plasma. Substitutionmutation of JBT0293 resulted in highly optimized peptides with respectto affinity for TFPI and improved activity in FXa inhibition, extrinsictenase inhibition, and plasma-based assays.

Example 5

The following example demonstrates that the peptides of the inventioncan be modified by the addition of moieties that enhance physicochemicalor pharmacokinetic properties of the peptides. As illustrated below, theaddition of 40 kDa PEG to peptides described herein dramaticallyimproved the pharmacokinetic behavior of the peptides. The example alsodescribes optimization of a TFPI-binding peptide, JBT1857, to reducesusceptibility to proteolysis.

Methods of conjugating chemical or biological moieties to peptides areknown in the art. To add PEG (polyethylene glycol) to the peptidesdescribe herein, a functional group (AOA=aminooxy acetate) was added tothe N-terminus of the peptides for coupling to aldehydes and ketones.Alternatively, a cysteine was added to the C-terminal part of thepeptide for coupling with maleimid (Hermanson, Bioconjugate Techniques,Academic Press (1996)). The peptides (JBT1586)AOA-FQSKGNVFVDGYFERL-Aib-AKL-NH2 (SEQ ID NO: 166) and (JBT1587)Ac-FQSKGNVFVDGYFERL-Aib-AKLC-NH2 (SEQ ID NO: 167) were used forN-terminal and C-terminal modification with PEG, respectively.AOA-FQSKGNVFVDGYFERL-Aib-AKL-NH2 (SEQ ID NO: 166) andAc-FQSKGNVFVDGYFERL-Aib-AKLC-NH2 (SEQ ID NO: 167) were incubated withexcess 40 kDa mPEG-Propionaldehyde (SUNBRIGHT ME-400AL2, NOF, Japan) and40 kDa mPEG-maleimide (SUNBRIGHT ME-400MA, NOF, Japan), respectively.The resulting PEGylated peptides, JBT1852 and JBT1855, show similaraffinities compared to the starting structureAc-FQSKGNVFVDGYFERL-Aib-AKL-NH1 (JBT0740) (SEQ ID NO: 66).

The resulting PEGylated peptides demonstrated significantly increasedplasma stability and prolonged plasma half-life in mice. FIG. 31illustrates the results from a pharmacokinetic analysis of the freepeptide JBT0740 (Ac-FQSKGNVFVDGYFERL-Aib-AKL-NH2) (SEQ ID NO: 66)compared to the C-terminally PEGylated peptide JBT1855(Ac-FQSKGNVFVDGYFERL-Aib-AKLC(PEG(40 kD))-NH2) (SEQ ID NO: 252)following intravenous administration to mice. In contrast to theunPEGylated peptide, the PEGylated peptide is present at highconcentrations in mouse plasma at 100 minutes post-administration. TheunPEGylated peptide is rapidly cleared from the plasma. FIG. 40illustrates the results from a pharmacokinetic analysis of JBT1855following subcutaneous injection. JBT1855 also strongly improvedthrombin generation in the assay described in Example 4 (FIG. 41).

The JBT1852 and JBT1855 peptides also were characterized in the assaysdescribed in Examples 1-4 and compared to JBT0740 and other peptides inthe JBT0047 family. Representative results are provided in Table 10 setforth below.

TABLE 10 FVIII- equivalent activity Solubility TFPI-1α FXa (mU/ml) in(mg/ml; Plasma Affinity Inhi- FVIII PBS Stability (nM) bition deficientwithout (half life in Biacore IC₅₀ plasma @ 1 Ca²⁺ and minutes) K_(D)(μM) μM peptide Mg²⁺) mouse human JBT0717 1.1 0.05 421   0.97  24 >120JBT0740 2.4 0.06 333   0.92  50 >120 JBT1584 0.3 0.02 486   2.66 40 >120 JBT1852 11.1 0.17 >1000 >1.00* >120 >120 JBT1855 10.50.07 >1000 >1.00* >120 >120 *formulated in 25 mm HEPES, pH 7.35, 175 mMNaCl

The peptides listed in Table 10 also were assayed for interaction withthe TFPI-2 anti-target, and generated signals too low for reliableaffinity measurement. The data suggest that PEGylation does not ablatethe inhibitory activity of the inventive peptides or negatively affectselectivity for TFPI-1.

Cell-Based Extrinsic Tenase Assay

The ability of the TFPI-binding peptides described above to restoreextrinsic tenase complex-mediated conversion of FX to FXa also wasdetermined using a cell-based extrinsic tenase assay. The cell-basedextrinsic tenase assay also was employed to explore the influence ofPEGylation on an exemplary TFPI-binding peptide of the invention,JBT0740. Human umbilical vein endothelial cells (HUVEC) were counted andseeded in complete growth medium in a 96-well plate (black flat withclear bottom) at a density of 1.5×10⁴ cells per well. Cells were grownovernight (for approximately 16 to 18 hours), washed twice withpre-warmed basal medium, stimulated with 1 ng/ml recombinant TNFα (SigmaAldrich (Cat. No. T6674)) in 200 μl of basal medium for four hours at37° C., and washed twice with 200 μl of pre warmed cell culture buffer.Buffer (50 μl) containing FVIIa (Enzyme Research Laboratories),TFPI-binding peptides (dissolved in either DMSO or Hepes buffered salinewith or without 0.1% Tween-80), or αTFPI antibodies were applied to thecells and incubated for 20 minutes at 37° C., allowing FVIIa/TF complexformation and binding of TFPI antagonists to TFPI. After the incubationperiod, 50 μl of cell culture buffer containing FX and a FXa-specificsubstrate (Fluophen FXa (HYPHEN BioMed)) was applied, resulting in afinal volume of 100 μl cell culture buffer mix on the cells. The finalconcentrations were: 39 pM FVIIa; 170 nM FX; 250 μM Fluophen FXa, and2.5% DMSO (when peptides were dissolved in DMSO).

The 96 well plate was transferred to a pre-warmed (37° C.) fluorescencereader for detecting FXa-specific fluorogenic substrate conversion byFXa, which is generated by the TF/FVIIa complex on the surface ofstimulated HUVECs. Readings taken after nine minutes of incubation wereused for calculation of the TFPI inhibitory effect of the TFPI-bindingpeptides or antibodies. The approximate percent inhibition of TFPIobserved at various concentrations of the following peptides (belongingto the JBT0047 family) is set forth in Table 11: JBT0717(Ac-FQSK-Nmg-NVFVDGYFERLRAKL-NH2) (SEQ ID NO: 61), JBT0740(Ac-FQSKGNVFVDGYFERL-Aib-AKL-NH2) (SEQ ID NO: 66), JBT1584(Ac-FQSK-Nmg-NVFVDGYFERL-Aib-AKL-NH2) (SEQ ID NO: 164), and JBT1857(Ac-FQSKpNVHVDGYFERL-Aib-AKL-NH2) (SEQ ID NO: 178).

TABLE 11 % TFPI inhibition 40 μM 8 μM 1.6 μM 0.32 μM 64 nM JBT0717 60%50% 32% 28% 20% JBT0740 70% 39% 28% 14%  3% JBT1584 73% 62% 51% 40% 29%JBT1857 80% 57% 41% 35% 15%

PEGylated peptides also were tested using the cell-based extrinsictenase assay. JBT0740 (SEQ ID NO: 66) was conjugated to a 1 kD PEGmoiety at the N-terminus to produce JBT1853 or at the C-terminus toproduce JBT1854. JBT1853 and JBT1854 inhibited TFPI by 20% or lessdepending on the amount of peptide used in the assay. JBT1855, whichcomprises a 40 kD PEG moiety at the C-terminus (parent peptide, JBT0740)performed better in the cell-based assay than JBT1852, which comprises a40 kD PEG moiety at the N-terminus. JBT1855 mediated 20-30% TFPIinhibition, while JBT1852 inhibited TFPI activity by 10% or less.

Peptides of the JBT0120 family, JBT0120, JBT0415, JBT0444, JBT1426, andJBT1837, also were tested in the cell-based extrinsic tenase assay andfound to inhibit TFPI to a lesser degree compared to peptides of theJBT0047 family. The reduced or partial inhibitory activity may bedesired in some embodiments of the invention. Similar to the peptides ofthe JBT0047 family, peptide optimization increased TFPI inhibitoractivity of JBT0120 family peptides.

In the course of examining the stability and inhibitory activity ofJBT1857, it was determined that the amino acid sequence of the peptidecontained a protease cleavage site between Val9 and Asp10. Substitutionof Tle at position 9 (generating JBT2431) and substitution of Pro atposition 10 (generating JBT2432) blocked cleavage of the peptide andenhanced the plasma stability of the peptide by about three-fold from27% (JBT1857) to 82% (JBT2431) and 76% (JBT2432). An additional putativecleavage site was identified between Gly11 and Tyr12. A G11asubstitution (generating JBT2414) further improved the stability of thepeptide to 100%. All stabilities were determined by quantitative ELISAafter 24 hour incubation in human plasma.

The results described above demonstrate that optimization of theTFPI-binding peptides described herein utilizing non-conventional aminoacids improved TFPI inhibition and plasma stability. Additionally,PEGylated peptides of the invention inhibit TFPI activity in acell-based extrinsic tenase assay, with C-terminal PEGylated peptidesperforming better than N-terminal PEGylated peptides. The TFPI-bindingpeptides of the invention inhibit the activity of both free TFPI andcell-bound TFPI.

Example 6

The following example illustrates the ability of peptides describedherein to reduce bleeding in an animal model.

Ten week old C57B1/6NCrl mice were housed for two weeks prior to thestudy. Thirty minutes before the nail clip, the animals wereadministered (a) JBT1855 (10 mg/kg) intravenously (i.v.) via the tailvein or subcutaneously (s.c.) in the neck region, (b) anti-TFPI antibody(18 mg/kg; i.v.), or (c) vehicle (175 mM NaCl, 25 mM HEPES, pH 7.35; 10ml/kg; i.v.). The animals were anaesthetized with 80 mg/kg pentobarbitalten minutes prior to the nail clip. To achieve bleeding, the nail of thesmall toe of the right hind paw was removed. The paw was submerged in a0.9% NaCl solution for blood collection for a period of 60 minutes.Blood loss was quantified after lysis by spectrophotometry. Thetemperature was kept constant at 37° C. over the course of theexperiment. The results of the study are illustrated in FIG. 42 andsummarized in Table 12.

TABLE 12 JBT1855 JBT1855 α-TFPI Vehicle i.v. s.c. i.v. i.v. Mean (in μl)29.9 31.7 1.9 74.9 (SD) (71.4) (31.5) (1.4) (74.6) # of mice 12 12 12 12p-value 0.07 0.04 0.001

Intravenous or subcutaneous administration of JBT1855, a PEGylatedpeptide of the invention, reduced blood loss in mice compared totreatment with vehicle alone.

Example 7

The following example describes characterization of TFPI-peptideinteractions via nuclear magnetic resonance and x-ray crystallography.In particular, the TFPI binding site of the antagonistic peptidesJBT0303, JBT0122 and JBT0415; the residues of JBT0303, JBT0122 andJBT0415 interacting with TFPI160; and the secondary structure ofcomplexed and free JBT0303, JBT0122 and JBT0415 were investigated at amolecular level using 2D ¹⁵N-heteronuclear single quantum coherence(HSQC) spectra. The interaction of JBT1857 and KD1 of TFPI was examinedusing x-ray crystallography, and the residues of TFPI KD1 that mediateJBT1857 binding were mapped.

Identification of the Binding Site of JBT0303 on TFPI160

A ¹⁵N-labelled preparation of TFPI160 was used for titration experimentsof TFPI160 with JBT0303. HSQC spectra of a ˜500 μM ¹⁵N-TFPI160 samplewithout and with increasing amounts of peptide were recorded at 30° C.on a Varian 600 MHz spectrometer. The peptide-protein interaction showedslow exchange behavior (k_(ex)<<Δω), meaning that each TFPI residueresults in a defined signal for the free protein and the protein-peptidecomplex. Unlike fast exchange behavior (k_(ex)>>Δω), where a mixtureresults in only one peak with averaged position according to thepopulation of the species, slow exchange behavior does not allowtracking of the signals upon peptide binding. Thus, in order to locatethe binding site, the shifted peaks of the TFPI160-JBT0303 complexneeded to be assigned. This required the preparation of a sample of¹³C/¹⁵N-TFPI160 and JBT0303.

Initially, a sample was prepared with 992 μM ¹³C/¹⁵N-TFPI160 and 1190 μMJBT0303. However, the NMR sample resulted in poor quality spectra whichdid not allow assignment of the complex. The sample gelled, likely dueto the formation of high molecular weight aggregates. Thus, the acquiredNMR data predominantly showed signals arising from the most flexibleparts of the isotope-labeled TFPI160. Therefore, sample conditions werereinvestigated for further experiments. From a series of ¹⁵N-HSQCexperiments conducted on the TFPI160-JBT0303 complex, it was concludedthat gel formation could be avoided by sample dilution and dataacquisition at elevated temperature. The final concentration of¹³C/¹⁵N-TFPI160 was 331 μM and that of JBT0303 was 397 μM. Spectraquality was improved. Due to the lower concentration and reducedsignal-to-noise ratio, assignment had to be performed based on HNCA,HNCO and HNCOCA experiments.

Except for four previously assigned residues, all residues that could beassigned in the apo-TFPI160 could be assigned in the TFPI160-JBT0303complex. Assignment of some residues was ambiguous due to the lack ofpeaks in the 3D spectra. Furthermore, the peaks of three residues wereonly visible in the HSQC spectrum from the original titrationexperiment. However, all peaks in the vicinity where unambiguouslyassigned and, therefore, the assignment of these residues is likely tobe correct.

Chemical shift changes of the HSQC signals of ¹⁵N-TFPI160 bound toJBT0303 compared to free TFPI160 is illustrated in FIG. 43. Residuesundergoing the strongest chemical shift were exclusively on Kunitzdomain 1. Chemical shifts of residues F25, F28, D32, A37, T48 and Y56shifted the most (>2 ppm). Residues I38, I46, F47 and F54 also shiftedmore than 1.5 ppm. It is unclear whether residues N-terminal of F25 areinvolved the interaction with JBT0303 because residues 20-24 are notassigned. L19 shows a change of chemical shift amounting to ˜0.6 ppm.Thus, in contrast to previous beliefs that amino acids within residues1-18 of TFPI are involved in peptide binding, the present data suggestthat there is little, if any, peptide binding to the N-terminal tail ofTFPI. A ribbon model of the secondary structure of TFPI proteinillustrating regions of chemical shift changes of HSQC signals ofTFPI160 bound to JBT0303 compared to free TFPI160 is set forth in FIG.44.

To more particularly identify the binding site of JBT0303 on TFPI160,the amide exchange rates of ¹⁵N-TFPI160 and ¹⁵N-TFPI160+JBT0303 weredetermined. The amide exchange experiment mainly detects changes in theenvironment of the peptide backbone by measuring H exchange of amidegroups. The H₂O frequency is irradiated with a power high enough that itis not dissipated by relaxation, resulting in a complete saturation andsuppression of the H₂O signal. A side effect of this method of H₂Osignal suppression is that the suppression is transferred toexchangeable amide NHs which exchange with solvent (H/H exchange). Thesaturation transfer is dependent on the H/H exchange rate which issemi-quantitative. The effect is reduced for more protected NH groups(i.e., unprotected NHs are attenuated more than protected NHs). If aprotected NH lies in proximity to H-alphas of a ligand, a higherexchange rate is observed compared to the apo form. Similarly, Hexchanges can be mediated by the OH groups of Ser, Thr or Tyr.

HSQC spectra without and with water suppression of apo ¹⁵N-TFPI160 andthe ¹⁵N-TFPI160-JBT0303 complex were recorded. The relative exchangerate of each residue of TFPI160 was determined by calculating the ratioof the peak intensities in the HSQC spectra with and without watersuppression. A comparison of the data sets of ¹⁵N-TFPI160 and¹⁵N-TFPI160+JBT0303 revealed that TFPI residues 25, 26, 36, 62, 63, 127,132 and 152 exhibited greater than 10% decreased amide exchange rate inthe complex, whereas residues 29, 30, 42, 45, 49, 50, 56, 66 and 98exhibited more than 10% increased exchange rate.

Constraints derived from the amide exchange experiment were included forthe calculation of refined HADDOCK models: (a) torsion angles are takenfrom the calculations of TALOS for K4, K5, V7, F8, Y12-A18 of JBT0303(chemical shift experiments); (b) residues of KD1 with chemical shiftchanges of more than 1.5 ppm are involved in binding JBT0303: F25, F28,D32, A37, I38, I46, F47, T48, F54 and Y56 (chemical shift experiments);(c) the hydrophobic side of the amphipathic helix of JBT0303 is bound toKD1: Y12 or L16 or L20 of JBT0303 bind to D32 or A37 or 138 or F54 orY56 of KD1 (chemical shift experiments); (d) R15 or K19 of JBT0303 bindto D31 or D32 or E60 of KD1 (chemical shift experiments); (e) F8 or V9of JBT0303 bind to F25 or F28 of KD1 (chemical shift experiments); (f)Y12 or F13 of JBT0303 bind to 146 or F47 or T48 of KD1 (chemical shiftexperiments); (g) Q2 of JBT0303 binds to Y56 of KD1 (chemical shiftexperiments); (h) F1 of JBT0303 binds to M39 or F66 of KD1 (amideexchange experiments); (i) S3 or K4 or K5 of JBT0303 bind to F66 (amideexchange experiments); (j) V7 or F8 or V9 of JBT0303 bind to F25 or C26or N62 or Q63 of KD1 (amide exchange experiments); (k) V9 or D10 or G11or R15 of JBT0303 bind to F28 or K29 or A30 of KD1 (amide exchangeexperiments); (l) Y12 or F13 of JBT0303 bind to N45 of KD1 (amideexchange experiments); (m) Y12 or F13 or E14 or R15 bind to R49 or Q50of KD1 (amide exchange experiments); and (n) L20 of JBT0303 binds to K36of KD1 (amide exchange experiments). The data converged to essentiallyone model of the KD1+JBT0303 complex.

Identification of the Binding Site of JBT0122 on TFPI160

As with the ¹³C/¹⁵N-TFPI160+JBT0303 complex, the ¹³C/¹⁵N-TFPI160+JBT0122NMR sample resulted in spectra of poor quality due to the formation of agel. The concentration of 723 μM ¹³C/¹⁵N-TFPI160+JBT0122 lead toformation of higher order aggregates. The sample was diluted to 361.5 μMand spectra recorded at 37° C., resulting in improved spectra quality.HNCO, HNCA and HNCOCA spectra were acquired. Except for five residues,all of the previously assigned peaks of apo-TFPI160 could be assigned inthe TFPI160-JBT0122 complex. Residues undergoing the strongest chemicalshift changes and likely to interact with the peptide often did notresult in peaks in the 3D spectra. Peaks in the linker region betweenKunitz domain 1 (KD1) and Kunitz domain 2 (KD2), however, also exhibitedlow intensities. Hence, the assignment of these peaks is ambiguous. Somepeaks were only visible in the HSQC of the original titrationexperiment. Their assignment was in most cases certain, as the peaksoverlapped in the TFPI160 and the TFPI160+JBT0122 HSCQ spectra.

Chemical shift changes of the HSQC signals of ¹⁵N-TFPI160 bound toJBT0122 compared to free TFPI160 is illustrated in FIG. 45. Significantchemical shift changes were exclusively found for residues of KD2. Ingeneral, the extent of the chemical shift changes caused by binding ofJBT0122 to TFPI160 was less pronounced than that of JBT0303. Residueswith the strongest perturbation of chemical shift were F96, G128, G129,G132, N133 and N136. C97, E101, T111, F114, N135 and F137 wereperturbed, exhibiting chemical shift changes of more than 0.5 ppm. Aribbon model of the secondary structure of TFPI protein illustratingregions of chemical shift changes of HSQC signals of TFPI160 bound toJBT0122 compared to free TFPI160 is set forth in FIG. 46.

Identification Residues of JBT0122 that Interact with TFPI160

For the sequential backbone signal assignment of JBT0122,¹³C/¹⁵N-labelled peptide was produced recombinantly. Briefly, thepeptide was expressed as a fusion protein with thioredoxin in E. coli.¹³C/¹⁵N-labelled peptide was prepared using M9 medium containing 3.0 g/1¹³C-glucose and 1.0 g/1 ¹⁵NH₄Cl. The fusion protein was affinitypurified using a Ni-chelating column and a poly-histidine tag. Thepeptide was cleaved by thrombin. The thioredoxin/his-tag and thrombinwas removed using a Ni-chelating column and a benzamidine column,respectively. The peptide was then purified by reverse phasechromatography. Purity, integrity, and identity were verified bySDS-PAGE, RP-HPLC and mass spectrometry. Recombinant JBT0122 was namedJBT0788 and had two additional residues at its N-terminus, glycine andserine, which represent the remains of the thrombin cleavage site.

The assignment of JBT0788 was done on the basis of HSQC, HNCACB, HNCA,HNCO and HNN spectra recorded at 10° C. on a Varian 600 MHz spectrometerand assigned using the SPARKY software. The temperature was reducedcompared to NMR experiments with TFPI160 to improve spectra quality.From the recorded spectra, the carbonyl carbon (C), the alpha carbon(CA), the beta carbon (CB), the amide proton (H), and the amide nitrogen(N) of most residues were assigned. The assignment for residues H13 andR17 was ambiguous. An HNCOCA led to an unambiguous assignment for theseresidues.

An assignment table for JBT0788 is provided in FIG. 47. Two sets ofsignals for residues 4-12 were observed in the spectra of JBT0788.Considering that the primary structure of JBT0788 is not compromised,the two sets of signals likely result from a cis/trans isomerization ofthe peptide bond between F6 and P7. A ratio of 76:24 was determined formajor:minor conformation based on the intensities of the correspondingsignals in the HSQC spectrum. As judged from the Cα shift of theproline, the major conformation is likely trans, as its Cα value of63.16 ppm is higher than of the minor conformation (62.49 ppm).

One purpose of the assignment was to extract the secondary structure ofthe peptide from Cα chemical shifts. Cα chemical shifts are influencedby the angles φ and ψ and, thus, by the secondary structure of thepeptide. In β-strands, Cα are generally shifted to lower ppm; inα-helices, Cα are generally shifted to higher ppm. By subtracting themeasured Cα value from a tabulated random coil value, negative valuesare calculated for residues in β-strands and positive values forresidues in α-helices. Thus, a batch of consecutive negative valuesindicates a α-strand while a batch of consecutive positive valuesindicates an α-helix.

JBT0788 exhibited a broad patch of increased Cα values(Δδ(Cα)=Cα_(measured)−Cα_(random coil)) indicating an α-helix comprisingresidues 8 to 26. Δδ(Cα) values for stable α-helices within tertiarystructures of native proteins are typically between 3-4 ppm. Δδ(Cα)values of the α-helix of JBT0788 rise up to about 1.7 ppm, indicatingmore flexibility than an average helix within a protein. Another featureof JBT0788 is the proline at position 7, directly N-terminal to theα-helix, which fits well as α-helices in proteins are frequentlyterminated by a proline at the N-terminus. Residue 6 has a strongnegative value, which is caused by the neighboring proline known toforce its N-terminal neighbor into a β-strand-like conformation. Thestrong positive value of C-terminal residue 31 is also typical forresidues without a C-terminal neighbor. The peptide bond between F6 andP7 in JBT0788 adopts two conformations, a trans (76%) and a cisconformation (24%). The conformation at this position impacts theconformation of the consecutive residues. In the trans isoform, theα-helix starts immediately after P7; the α-helix of the cis isoform doesnot start until residue L12. A ribbon model illustrating the secondarystructure of free JBT0788 is set forth in FIG. 48.

The chemical shifts within JBT0788 can also be employed to calculate thetorsion angles using TALOS software. TALOS is a database system forempirical prediction of φ and ψ backbone torsion angles using acombination of five kinds (HA, CA, CB, CO, N) of chemical shiftassignments for a given protein or peptide sequence. The TALOS approachis an extension of the observation that many kinds of secondary chemicalshifts (i.e., differences between chemical shifts and theircorresponding random coil values) are correlated with aspects of proteinsecondary structure. The goal of TALOS is to use secondary shift andsequence information in order to make quantitative predictions for theprotein backbone angles φ and ψ, and to provide a measure of theuncertainties in these predictions. TALOS uses the secondary shifts of agiven residue to predict φ and ψ angles for that residue. TALOS alsoincludes the information from the next and previous residues when makingpredictions for a given residue. The idea behind TALOS is that if onecan find a triplet of residues in a protein of known structure withsimilar secondary shifts and sequence to a triplet in a target protein,then the φ and ψ angles in the known structure will be useful predictorsfor the angles in the target. In practice, TALOS searches a database forthe 10 best matches to a given triplet in the target protein.

In order to assign the HSQC spectrum of JBT0788 complexed with TFPI160,a sample consisting of 400 μM ¹³C/¹⁵N-JBT0788 and 400 μM TFPI160 wasprepared. As with previous NMR samples of peptide and TFPI160, thesample gelled. The sample was diluted and the pellet dissolved indeuterated DMSO, resulting in a final concentration of ˜300 μM¹³C/¹⁵N-JBT0788+TFPI160 and 5% DMSO. Measurements were performed at 40°C. This improved the quality of the acquired spectra. Experiments wereacquired in the TROSY mode to account for the relaxation properties of apartially aggregated sample. Cryo-probe technology on the Varian 600 MHzspectrometer was employed due to the low concentration of theprotein-peptide complex in the sample. The resulting data quality wassufficient to obtain the backbone shifts of JBT0788 when utilizing thecryo-probe technology and acquiring the triple-resonance experiments induplicate. The assignment of JBT0788 in complex with TFPI160 wasperformed on the basis of HNCA, HNCOCA and HNCO spectra. From therecorded spectra, the carbonyl carbon (CO), the alpha carbon (CA), theamide proton (H), and the amide nitrogen (N) of most residues wereassigned. An assignment table for JBT0788 complexed to TFPI160 isprovided in FIG. 49.

A feature of apo-JBT0788 was the presence of two sets of signals foramino acid residues 4-12, likely resulting from a cis/transisomerization of the peptide bond between F6 and P7. In theJBT0788-TFPI160 complex, only one set of peaks is observed, implyingthat only one of the conformations binds to TFPI160. Apo-JBT0788 alsoexhibited a broad patch of increased Cα values(Δδ(Cα)=Cα_(measured)−Cα_(random coil)=positive) indicating an α-helixreaching from residue 8 to residue 26. As mentioned above, Δδ(Cα) valuesfor stable α-helices within tertiary structures of native proteins aretypically between 3-4 ppm. Δδ(Cα) values of the α-helix of apo-JBT0788increase to about 1.7 ppm, indicating more flexibility than an averagehelix within a protein. When complexed with TFPI, residues 8 to 26exhibited values of between 3-5 ppm, indicating the formation of astable α-helix or helices. A ribbon model illustrating the secondarystructure of JBT0788 when complexed with TFPI160 is set forth in FIG.50. Large chemical shift changes within JBT0788 caused by binding withTFPI160 are evenly distributed over the length of the peptide. Residuesundergoing the strongest perturbation of chemical shift were residuesS5, A9, Q11, Y28, and K29 with more than 4 ppm. Residues Y3, A4, V10,L12, S15, M21, A22, L23, and A24 were perturbed by more than 3 ppm.

Identification of Residues of JBT0303 that Interact with TFPI160

JBT0303 was produced recombinantly using the same procedure as describedabove for JBT0122 and isotope-labeled with ¹³C and ¹⁵N. The recombinantJBT0303 was named JBT0616 and had an additional glycine and serine atits N-terminus. The assignment of JBT0616 was performed on the basis ofHSQC, HNCACB and HNN spectra, which were recorded at 10° C. on a Varian500 MHz spectrometer and assigned using SPARKY software. The quality ofthe spectra of JBT0616 was better than that of JBT0788, although theexperimental conditions with respect to buffer, temperature, NMR tube,and NMR parameters were identical. The alpha carbon (CA), the betacarbon (CB), the amide proton (H), and the amide nitrogen (N) of mostresidues were assigned. The assignment was mainly based on the lesssensitive but more informative HNCACB instead of the HNCA. Incombination with the HNN spectrum, this resulted in an unambiguousassignment of all JBT0303 derived residues.

An assignment table for JBT0616 is provided in FIG. 51. The secondarystructure was extracted from Cα chemical shifts and determined by TALOSusing the assignments of H, CA, CB, CO and N. Like JBT0788, JBT0616exhibited a patch of positive Δδ(Cα) values indicative of α-helicalconformation. The helix was located at the C-terminal part of thepeptide and comprised residues 10-18. As for JBT0788, Δδ(Cα) values upto about 1.8 ppm were calculated, qualifying this helix as relativelystable for such a short peptide. A ribbon model illustrating thesecondary structure of JBT0616 is set forth in FIG. 52. The strongpositive value of the C-terminal residue 20 is, like residue 31 inJBT0788, typical for residues without a C-terminal neighbor. TheN-terminal residues 1-9 of JBT0616 exhibited slightly positive Δγ(Cα)values, suggesting a preference for an α-helical structure.

The assignment of JBT0616 in complex with TFPI160 was performed using a¹³C/¹⁵N-labelled peptide sample with an excess of unlabelled TFPI160.HSQC, HNCA, HNCOCA, and HNCO spectra were recorded on a Varian 800 MHzspectrometer and assigned using the SPARKY software. The spectra wererecorded at 30° C. Using these spectra, the alpha carbon (CA), the betacarbon (CO), the amide proton (H), and the amide nitrogen (N) of mostresidues were assigned, as set forth in the table in FIG. 53. Thesecondary structure of JBT0616 in complex with TFPI160 was extractedfrom Cα chemical shifts and calculated by TALOS Like the free peptide,JBT0616 in complex with TFPI160 exhibited a C-terminal patch of positiveΔδ(Cα) values indicative of α-helical conformation. The stability of theα-helix is increased upon complex formation. This finding suggests thatthe C-terminal region of JBT0616 is the core binding motif. The Δδ(Cα)values for the N-terminal residues also changed, but to a lesser extent.The secondary structure of JBT0616 when complexed with TFPI isillustrated in the ribbon model in FIG. 54.

The most significant changes of chemical shifts upon complex formationwere observed for residues Q2, K5, F8, V9 and A18 of JBT0616 with morethan 7 ppm. Residues F13, R17, K19 and L20 also were perturbed anddemonstrated chemical shift changes of more than 4 ppm. The strongchemical shift changes of residues at the N-terminus indicated that itis not only the amphipathic C-terminal α-helix which drives binding ofthe peptide to TFPI160.

Results from the NMR experiments in combination with analysis of JBT0477substitutions were used to create a model of KD1 in complex with JBT0303using HADDOCK (High Ambiguity Driven protein-protein DOCKing) software.HADDOCK is an information-driven flexible docking approach for themodeling of biomolecular complexes. HADDOCK distinguishes itself fromab-initio docking methods in the fact that it encodes information fromidentified or predicted protein interfaces in ambiguous interactionrestraints (AIRs) to drive the docking process. Identification of thebinding site on TFPI160 and the peptides as revealed by chemical shiftdata, the torsion angles of the peptides as determined by the softwareTALOS, and the substitution analysis of JBT0477 provide the restraintsfor the calculation of the models.

For the calculation of the KD1-JBT0303 HADDOCK models, the followingrestraints were employed: (a) torsion angles were taken from thecalculations of TALOS for K4, K5, V7, F8, Y12-A18 of JBT0303; (b)residues of KD1 with chemical shift changes of more than 1.5 ppm areinvolved in binding to JBT0303: F25, F28, D32, A37, I38, I46, F47, T48,F54 and Y56; (c) the hydrophobic side of the amphipathic helix ofJBT0303 is bound to KD1: Y12 or L16 or L20 of JBT0303 bind to D32 or A37or 138 or F54 or Y56 of KD1; (d) R15 or K19 of JBT0303 binds to D31 orD32 or E60 of KD1; (e) F8 or V9 of JBT0303 binds to F25 or F28 of KD1;(f) Y12 or F13 of JBT0303 binds to 146 or F47 or T48 of KD1; and (g) Q2of JBT0303 binds to Y56 of KD1. The Q2 JBT0303-Y56 KD1 interaction alsowas taken as a restraint for model calculation.

Strong chemical shift changes were observed for K5 of JBT0303 uponcomplex formation. For the remaining residues of JBT0303 considered todrive the peptide-protein interaction, the models are in good agreementwith the data. The model of KD1-JBT0303 with the lowest energy places F8of JBT0303 in proximity to F25 and F28 of TFPI, explaining the observedchemical shift changes and the data from the substitution analysis. V9of JBT0303 interacts with the hydrophobic patch of the KD1 includingF54. Y12, F13, L16 and L20 of JBT0303 also face the hydrophobic patch ofthe KD1. The proximity of Y12 to F28, I46, T48 of F13 to F47, T48, ofL16 to F54 and of L20 to A37, I38 causes the observed perturbations ofNMR chemical shift of those residues in the complex; the conservation ofY12 and L16 may be due to the extensive interactions of these residueswith the protein. K19 of JBT0303 is in a position allowing interactionwith D32 of KD1. The role of R15 of JBT0303 seems to be an interactionwith the hydrophobic patch of KD1 as well as with D32. Moreover, themodel explains why a negatively charged aspartate is preferred atposition 10 of JBT0303; it can interact with the positively charged K29of KD1. A glycine at position 11 of JBT0303 is present due to the stericand conformational restraints at this position. A HADDOCK model of KD1(TFPI residues 22-79 comprising KD1) in complex with JBT0303 is providedin FIG. 55.

Models of JBT0740 and JBT1857 Bound to KD1

Peptides JBT0740 and JBT1857 (FQSK-dP-NBHBDGYFERL-Aib-AKL (SEQ ID NO:178)), both derivatives of JBT0303, demonstrate significantly enhancedEC₅₀ values in the FXa-TFPI inhibition assay (0.11 μM and 0.0023 μM,respectively) and lower K_(d)'s as determined by Biacore. Models ofJBT0740 and JBT1857 in complex with TFPI KD1 (residues 22-79 of TFPI160)were calculated by HADDOCK using similar constraints as for JBT0303: (a)the constraints for the torsion angles of residues 4 and 5 of JBT0740and JBT1857 were amended in order to take account of the substitutionsat position 5 of the JBT0303 derivatives; (b) torsion angles were takenfrom the calculations of TALOS for V7, F8, Y12-A18 of JBT0303 and, incontrast to JBT0303, no fixed values for Phi and Psi were given for K4and for NmetGS/dPS; (c) NmetG5 and dP5 are in the cis conformation; (d)residues of KD1 with chemical shift changes of more than 1.5 ppm areinvolved in binding to JBT0303: F25, F28, D32, A37, I38, I46, F47, T48,F54 and Y56; (e) the hydrophobic side of the amphipathic helix ofJBT0303 is bound to KD1; (f) Y12 or L16 or L20 of JBT0303 bind to D32 orA37 or 138 or F54 or Y56 of KD1; (g) residues R15 or K19 of JBT0303 bindto D31 or D32 or E60 of KD1; (h) residues F8 or V9 of JBT0303 bind toF25 or F28 of KD1; (i) residues Y12 or F13 of JBT0303 bind to 146 or F47or T48 of KD1; and (j) residue Q2 of JBT0303 binds to Y56 of KD1.

The energetically most favorable HADDOCK models of JBT0740 and JBT1857illustrated a different mode of binding compared to JBT0303. The mostobvious differences were in the region of residues 5 to 11. Lessdramatic deviations were observed at the N-terminus and the C-terminusof the peptides. However, the different binding of the termini alsomight contribute to the optimized binding of the JBT0303 derivatives toTFPI.

X-Ray Crystal Structure of JBT1857 Bound to KD1

The crystal structure of KD1 in complex with a KD1 binding peptide,JBT1857, was determined. TFPI was recombinantly expressed in E. coli andoxidatively refolded from inclusion bodies. TFPI amino acids 1-150comprising a thrombin cleavage site within the TFPI linker sequencejoining KD1 and KD2 (TFPI1-150-Thrombin(MADSEEDEEHTIITDTELPPLKLMHSFCAFKADDGPCKAIMKRFFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRDNANRLVPRGSQQEKPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNICEDG (SEQ ID NO: 4235)) was clonedinto an E. coli expression vector (pET19b). The TFPI 1-150-Thrombinsequence comprises two amino acids at the N-terminus that are artifactsof recombinant expression, and are not part of the wild-type TFPI aminoacid sequence. The sequences encoding Kunitz domain 1 and 2 are bolded.E. coli (BL21(DE3) pLysS) was cultivated in MagicMedia™ and TFPI1-150-Thrombin was expressed as insoluble inclusion bodies. Inclusionbodies were harvested by lysis of E. coli by incubation with BugBusterMaster Mix and purified upon washing with 50 mM Tris/HCl pH 8, 0.1%Tween 20. Inclusion bodies were dissolved in 8M urea, 50 mM Tris/HCl pH8.0 and TFPI 1-150-Thrombin was reduced upon addition of 20 mM DTT.Oxidative refolding was performed by rapid 1/10 dilution into a buffercontaining 50 mM Tris/HCl pH 10 and 1.1 mM oxidized Glutathion, followedby excessive dialysis against 20 mM Tris/HCl pH 7. RefoldedTFPI1-150-Thrombin was purified by a sequential purification protocolusing a Q Sepharose FF anion exchange and a peptide affinity (JBT131)media. Purified TFPI1-150-TFPI was proteolytically digested byincubation with thrombin (1 U thrombin/mg TFPI1-150-Thrombin, cleavagesite, LVPR/GS) resulting in the generation of Nterm KD1-Thrombin(MADSEEDEEHTIITDTELPPLKLMHSFCAFKADDGPCKAIMKRFFFNIFTRQCEEFIGGCEGNQNRFESLEECKKMCTRDNANRLVPR (SEQ ID NO: 4236)) and KD2-Thrombin(GSQQEKPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLE ECKNICEDG (SEQID NO: 4237)). Nterm KD1-Thrombin was purified from the digestionmixture using benzamidin sepharose for removal of thrombin, followed bya JBT131 peptide affinity column. Purified Nterm KD1-Thrombin was usedfor complex formation with JBT1857 and further crystallization.

The antagonistic peptide, JBT1857, was prepared by solid phasesynthesis. Successful co-crystallization of equimolar complexes wasobtained under 100 mM MES pH 6.5, 20% PEG 4000, 600 mM NaCl. Crystalsdiffracted to better than 2.5 Å resolution, albeit with somenon-merohedral twinning. Diffraction data were processed with iMosflmand SCALA from the CCP4 program package, revealing a monoclinic crystalform with unit cells dimensions of a=113.67 Å, b=69.32 Å, c=42.37 Å,α=90.0°, β=92.97°, γ=90.0°, spacegroup C2 (Leslie, Acta Crystallogr DBiol Crystallogr, 62(Pt 1), 48-57 (2006); Evans, Acta Crystallogr D BiolCrystallogr, 62(Pt 1), 72-82 (2006)). Self-rotation calculationsindicated an approximately two-fold non-crystallographic symmetry.Consistent herewith, two molecules were localized in the asymmetric unitrelated by a 170° rotation. The Patterson search was carried out byusing the program PHASER and a structure ensemble of the availableKunitz domain 2 crystal structures as search model (McCoy et al., J ApplCrystallogr, 40(Pt 4), 658-674 (2007)). The unit cell containedapproximately 64% solvent. Non-crystallographic electron densityaveraging and model building and model refinement was carried out withCoot, Refmac, MAIN and CNS programs. The current model was completelydefined for both copies of the JBT1857 peptide and the interaction withthe protein with current R=0.257, Rfree=0.298, deviation from idealgeometry rms(bond)=0.008 Å, rms(angle)=1.8°.

JBT1857 Structure:

The structure of JBT1857 can be segmented into (i) the N-terminal anchorconsisting of acetylated Phe1_(AP)-Gln2_(AP); (ii) an Ω-shaped loopcomprising Ser3_(AP)-Asn6_(AP); (iii) an intermediate segment built fromVal7_(AP) and His8_(AP); (iv) a tight glycine-loop containingVal9_(AP)-Gly11_(AP); and (v) the C-terminal α-helix comprisingTyr12_(AP)-Leu20_(AP). As used herein, the subscript _(AP) indicates thesequence numbering in the “antagonistic peptide” JBT1857. Theconformation of the α-helix is stabilized by a non-natural α-methylalanine positioned at the center of the helix (position 17_(AP)); aC-terminal amide that completes the 1-4 hydrogen bonding pattern of theα-helix; and a stacked cluster by the aromatic side chains of His8_(AP),Tyr12_(AP) and Phe13_(AP). These effects cooperate to stabilize theC-terminal α-helix spontaneously in solution, consistent with circulardichroism data on the peptide. The observed aromatic side chain stacking(His8_(AP), Tyr12_(AP), Phe13_(AP)) enforces a tight turn that can beonly accomplished by glycine at position 11_(AP). This structuralconstraint is reflected by dramatic losses in binding affinity uponreplacement of Gly11_(AP) by any other amino acid. The conformation ofthe N-terminal loop segment is partly stabilized by a D-proline, knownto induce a tight turn conformation, and a 1-4 hydrogen bond by thecarbonyl oxygen of Ser3 with the amide nitrogen of Asn6_(AP). All ringside chains (Tyr1_(AP), Pro5_(AP), His8_(AP), Tyr12_(AP), Phe13_(AP))point towards the same direction, enabling them to interact with the KD1domain of TFPI.

Interaction of JBT1857 and KD1:

The interactions between JBT1857 and KD1 were determined. Hydrophobiccontacts are interactions having an intermolecular distance of ≦4 Å,while hydrogen bonds have a distance between 2.6-3.2 Å. Phe1_(AP)interacts non-specifically with TFPI making contacts with Phe2 andAla27. In contrast, Gln2_(AP) contacts a deeply buried pocket of TFPIand makes hydrophobic interactions with Phe28, Lys29, Ile46 and Phe47.Moreover, the amide group of Gln2_(AP) forms three H-bonds withPhe28-CO, Phe44-CO and Ile46-NH. The Ω-loop of JBT1857, comprisingSer3_(AP)-Asn6_(AP), mediates rather limited hydrophobic interactionswith the protein; Ser3_(AP), Pro5_(AP) and Asn6_(AP) interact with Lys29and Phe47. Val7_(AP) of JBT1857's intermediate segment also binds toLys29 and Phe47. His8_(AP) mainly contributes intramolecular aromaticstacking interactions with Tyr12_(AP) and partly with Phe13_(AP), andexhibits a hydrophobic interaction with Ala30 of TFPI. Similarly, theglycine-loop Val9_(AP)-Gly11_(AP) contributes few contacts with theKunitz domain. Val9_(AP) interacts directly with KD1 by forming ahydrogen bond with the carbonyl group of Ala30 and a hydrophobicinteraction with Asp32. Tyr12_(AP) mediates a hydrogen bond via itshydroxyl group with the amide nitrogen of Ile55 and a hydrophobicinteraction with Asp30. Leu16_(AP) is part of a hydrophobic contact withIle55. Beside the largely hydrophobic interactions of the C-terminalhelix of the peptide with the protein, there are electrostaticinteractions between Arg15_(AP) and Asp32. Furthermore, Lys19_(AP)contributes to binding with TFPI by forming a hydrogen bond to thecarbonyl group of Ala37 and contacts with Lys36 and Ile38. The TFPIcontact surface has an overall hydrophobic character with some chartedhot spots, and a driving force of complex formation with JBT1857 is thesteric surface complementarity.

This example describes characterization of the secondary structure ofexemplary peptides of the invention and correlates the structure withinhibitory function of the peptides. The example also identifies theTFPI amino acid residues that interact with JBT1857, a TFPI-bindingpeptide that inhibits TFPI activity.

Example 8

The following example describes additional TFPI-binding peptidesmodified by the addition of moieties that enhance physicochemical orpharmacokinetic properties of the peptides. The example furtherdescribes a method for assessing clot formation in whole blood usingrotation thromboelastography.

JBT1857 (JBT0047 peptide family) was conjugated to different PEGmoieties, and the binding affinity and TFPI inhibitory activity of thePEGylated peptides were examined. JBT1857 was modified by addition of aC-terminal cysteine to produce JBT2315 (Ac-FQSKpNVHVDGYFERL-Aib-AKLC-NH2(SEQ ID NO: 4077)), which was conjugated at the C-terminus with linearmaleimide PEG moieties of increasing size: 5 kD, 12 kD, 20 kD, 30 kD,and 40 kD, using the methods described in Example 5. The resultingPEGylated peptides were designated as follows:

TABLE 13 SEQ PEG ID Peptide (kD) Sequence NO JBT1857 —Ac-FQSKpNVHVDGYFERL-Aib-AKL-NH2 4020 JBT2317 —Ac-FQSKpNVHVDGYFERL-Aib-AKLC 4078 (NEM)-NH2 JBT2325  5.3Ac-FQSKpNVHVDGYFERL-Aib-AKLC 4086 (PEG)-NH2 JBT2326 12.1Ac-FQSKpNVHVDGYFERL-Aib-AKLC 4087 (PEG)-NH2 JBT2327 21.0Ac-FQSKpNVHVDGYFERL-Aib-AKLC 4088 (PEG)-NH2 JBT2328 29.1Ac-FQSKpNVHVDGYFERL-Aib-AKLC 4089 (PEG)-NH2 JBT2329 41.5Ac-FQSKpNVHVDGYFERL-Aib-AKLC 4090 (PEG)-NH2Stability, Binding Affinity, and TFPI-Inhibitory Activity of PEGylatedPeptides

The PEGylated peptides demonstrated significantly increased plasmastability in mouse and human plasma. The peptides were added to samplesof mouse or human plasma, and the percentage of the initial amount ofpeptide remaining in plasma 24 hours after the addition was measured byIC₅₀ ELISA on Maxisorp plates coated with 0.05 mg/ml TFPI (2.26 nMtracer peptide JBT2271). Less than approximately 10% of the initialamount of JBT1857 and JBT2317 remained in plasma, while 40% or more ofthe initial amount of the PEGylated TFPI-binding peptides remained after24 hours. Approximately 60% or more of JBT2327 and JBT2329 was detected.PEGylated peptides also are significantly more stable in human plasmacompared to unmodified peptides. Approximately 60% or more of PEGylatedpeptide remained after 24 hours. The unmodified peptides were morestable in human plasma than mouse plasma; about 20% or more of theinitial amount remained after 24 hours of incubation.

The PEGylated peptides also were characterized in the assays describedin Examples 1-4 and compared to JBT1857. Representative results areprovided in Table 14 set forth below. The thrombin generation assay wasperformed as described in Example 4, and the results are provided asEC₅₀, corresponding to the concentration of peptide which improved peakthrombin (nM) half maximal.

TABLE 14 Thrombin generation in human Compe- Extrinsic FVIII- tition FXaTenase inhibited Biacore ELISA Inhibition Inhibition plasma PEG K_(D)IC₅₀ EC₅₀ EC₅₀ EC₅₀ (kD) (nM) (nM) (nM) (nM) (nM) JBT1857 0.061 3.0 3.76.9 JBT2317 0.054 2.9 3.8 7.8 88 JBT2325 5.3 0.71 6.6 10.7 10.7 35JBT2326 12.1 1.1 9.3 9.3 9.3 34 JBT2327 21.0 1.3 10.9 7.2 7.2 24 JBT232829.1 1.6 12.3 6.0 6.0 19 JBT2329 41.5 1.1 12.6 6.0 12.8 19 *CompetitionELISA performed with tracer JBT2271 (1 nM) and 0.05 μg/ml TFPI in thecoating buffer.

Addition of the C-terminal cysteine blocked with NEM did notsignificantly influence the binding affinity of JBT2317 or the activityof the peptide in the FXa inhibition, extrinsic tenase assay, orthrombin generation assay compared to JBT1857. PEG size did notsignificantly impact the TFPI-binding peptides' ability to restoreactivity of FXa in the presence of TFPI-1. In the extrinsic tenase assayof Example 3, inhibitory activity increased with higher molecular weightPEG moieties up to 20 kD PEG. Activity did not further improve for 30 kDor 40 kD PEG moieties. In the thrombin generation assay of Example 4using human plasma, EC₅₀ decreased with PEG size, and maximal inhibitionof TFPI (as measured by peak FIIa (nM)) increased with PEG size. Inmouse plasma, attachment of 40 kD PEG to a TFPI binding peptideincreased maximal inhibition of TFPI.

The ability of PEGylated TFPI-binding peptides to restore extrinsictenase complex activity for converting FX to FXa also was determinedusing a cell-based extrinsic tenase assay using the method of Example 5.Addition of the C-terminal cysteine blocked with NEM did notsignificantly influence the activity of JBT2317 in the cell-basedextrinsic tenase assay compared to JBT1857. Conjugation of PEG moieties(5 kD, 20 kD, 30 kD, or 40 kD) to JBT2317 increased TFPI inhibitoryactivity by 5-20%.

Rotational Thromboelastography

Continuous visco-elastic assessment of human whole blood clot formationand firmness was performed by rotation thromboelastography with wholeblood preparations in the presence or absence of peptides. Blood samplesfrom a healthy individual were drawn into citrated Sarstedt Mono S(0.106 M or 3.2% (w/v) Na-citrate) (5 ml), mixing one part of citratewith nine parts blood, using a 21 gauge butterfly needle. A portion ofthe blood samples was incubated with high titer, heat-inactivatedanti-human FVIII antiserum raised in goat (3876 BU/ml; BaxterBioScience, Vienna, Austria) resulting in 51 BU/mL. Test samples wereprepared by dissolving quantities of peptides in either DMSO or HEPESbuffered saline (with or without 0.1% Tween 80).

Recordings were made using a ROTEM thromboelastography coagulationanalyzer (Pentapharm, Munich, Germany) at 37° C. Briefly, blood is addedinto a disposable cuvette in a heated cuvette holder. A disposable pin(sensor) is fixed on the tip of a rotating axis. The axis is guided by ahigh precision ball bearing system and rotates back and forth. The axisis connected with a spring for the measurement of elasticity. The exactposition of the axis is detected by the reflection of light on a smallmirror on the axis. The loss of elasticity when the sample clots leadsto a change in the rotation of the axis. The data obtained are computeranalyzed and visualized in a thromboelastogram. The thromboelastogramshows elasticity (mm) versus time (s). An elasticity of approximatelyzero is observed before clot formation begins. Mirror image traces aboveand below the zero line indicate the effect of clot formation on therotation of the axis.

Before starting each experiment, the citrated whole blood was mixed withcorn trypsin inhibitor (CTI) (Hematologic Technologies, Inc., EssexJunction, Vt., USA) providing a final concentration 62 μg/mL forspecific inhibition of FXIIa, in order to inhibit FXIIa-mediated contactactivation. The analytical set-up was as follows: to 20 μL of testsample or control, 300 μL of pre-warmed (37° C.) CTI treated citratedwhole blood was added, followed by 20 μL of a 1:15 dilution of TF PRPreagent containing recombinant human tissue factor (rTF, 3 pM) (TS40,Thrombinoscope BV, Maastricht, The Netherlands). Coagulation wasinitiated by the addition of 20 μL 200 mM CaCl₂ (Star-TEM®, Pentapharm,Munich, Germany) and recordings were allowed to proceed for at least 120min. The final concentration of rTF in the assay was 11 or 44 fM.

The thromboelastographic parameters of clotting time (CT), clotformation time (CFT) and maximum clot firmness (MCF) were recorded inaccordance with the manufacturer's instructions. CT is defined as thetime from the start of measurement to the start of clot formation. CFTis defined as the time from the start of clot formation until anamplitude of 20 mm is reached. MCF is the maximum difference inamplitude between the two traces during the assay. The first derivativeof the data of the thromboelastogram are plotted to obtain a graph ofvelocity (mm/s) against time (s). From this graph, the maximum velocity(maxV) is determined. The time at which the maximum velocity is obtained(maxV-t) is also determined.

Exemplary results are illustrated in FIGS. 56 and 57. JBT1857 andJBT2317 restored coagulation parameters in Hem A blood. PEGylated (40kD) TFPI-binding peptide JBT2329 also restored prolonged coagulationparameters in Hem A blood, as illustrated in FIG. 57. PEGylation ofJBT2317 reduces clot time and clot formation time.

Nail Clip Study

The effect of JBT2329 on blood loss in naïve mice also was studied.C57BL6 mice were administered vehicle, 1 mg/kg JBT2329, or 0.1 mg/kgJBT2329 (N=19 or 20 for each group) intravenously 30 minutes prior tonail clip at 10 ml/kg. Animals were anaesthetized 10 minutes before nailclip with 80 mg/kg pentobarbital (i.p.). At time=0 minutes, the nail ofthe small toe of the right hind paw was cut just before the nail bed.The paw was transferred to a vial prefilled with 0.9% NaCl solution.Samples of blood were collected for analysis during the first 30 minutesfollowing the nail clip and the next 30 minutes thereafter, and meancollected volume for the groups was calculated and compared. Mean bloodloss in vehicle treated mice was about 30.5 μl over the first 30minutes, 52.1 over the second 30 minute period, resulting in about 82.6μl of blood loss over 60 minutes. In contrast, administration of 0.1mg/kg JBT2329 reduced blood loss by about 50% over the first 30 minutes(16.0 μl) and about 64% over the second 30 minute period (18.7 μl),resulting in about a 60% reduction of total blood loss over 60 minutes(34.7 μl) compared to vehicle-treated mice. Increasing the dose ofJBT2329 to 1.0 mg/kg further reduced blood loss by at least about 10%;12.2 μl was collected over the first 30 minutes, 10.6 μl was collectedover the second 30 minute period, resulting in 22.8 μl collected overthe entire 60 minute collection period. JBT2329 also efficiently reducedbleeding when administered subcutaneously compared to vehicle-treatednaïve mice; subcutaneous injection of 10 mg/kg JBT2329 reduced bloodloss during the 60 minutes following nail clip by approximately 58%compared to vehicle-treated subjects.

The results described above were generated using a JBT1857 derivativecomprising a linear PEG moiety attached to the C-terminus of the peptideand a JBT1586 derivative comprising a PEG moiety at the N-terminus.Peptides comprising an alternate conjugation site or alternativechemical moiety also were generated. A 40 kD linear PEG moiety wasconjugated to residue 14 of JBT1857 to generate JBT2404. The linear 40kD PEG moiety of JBT2329 was replaced with a 40 kD branched PEG moietyto generate JBT2401. JBT1857 also was modified to compriseK(Ttds-Maleimidopropionyl) (JBT2374) at the C-terminus. JBT2374 was usedto generate JBT2410, an HSA conjugate of JBT2374. JBT2375, aK(AOA)-comprising derivative of JBT1857, was used to couple PSA aldehydeto the peptide JBT1857, resulting in JBT2430. JBT2401, JBT2404, JBT2410and JBT2430 were characterized using the assays described above.Representative results are summarized in Table 15:

TABLE 15 Thrombin generation in human Human FVIII- ELISA FXa Plasmainhibited Biacore affinity Inhibition Stability, 24 plasma K_(D) EC₅₀EC₅₀ hour EC₅₀ (nM) (nM) (nM) (%) (nM) JBT2329 <1 12.6 6 67.3 1.4JBT2401 <1 22.4 7.7 85.1 1.4 JBT2404 <1 18.2 13.4 96.7 1.7 JBT2410 n.d.5.1 4.7 65.7 1.8 JBT2430 n.d. 5.6 9.0 135.6

This example demonstrates that an exemplary TFPI-binding peptide of theinvention, JBT1857, is a potent inhibitor of TFPI and can befunctionalized and conjugated with PEG without loss of activity.PEGylation increased TFPI-inhibitory activity in several functionalassays. Surprisingly, peptides conjugated to higher weight PEG moietiesdemonstrated enhanced TFPI inhibitory activity. JBT2329, comprising a 40kD linear PEG moiety, significantly reduced blood loss in aclinically-relevant animal model. PEG conjugation within the amino acidsequence of JBT1857, use of a branched PEG moiety, and attachment of HSAand PSA did not destroy the activity of the peptide.

Example 9

The following example describes the characterization of two TFPI-bindingpeptides of the invention, JBT1837 and JBT1857. JBT1837(Ac-SYYKWH[CAMRDMKGTMTC]VWVKF-NH) (SEQ ID NO: 1044) is a cyclic peptideof the JBT0120 family that binds KD1 and KD2 of TFPI. JBT1857(Ac-FQSKpNVHVDGYFERL-Aib-AKL-NH2) (SEQ ID NO: 178) is a linear peptideof the JBT0047 family that binds KD1 of TFPI. The affinity andTFPI-inhibitory activity of JBT1837 and JBT1857 were examined using theassays described in Examples 1-4, the results of which are summarized inTable 16.

TABLE 16 Extrinsic Thrombin Thrombin FXa Tenase generation generationInhibition; Inhibition; in human in human ELISA R&D R&D FVIII-inhibitedFIX-deficient Biacore affinity TFPI/flTFPI TFPI/flTFPI plasma plasmaK_(D) EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ (nM) (nM) (nM) (μM) (nM) (nM) JBT1837 <14.8 3.2/5.9 0.5/0.9 10 16 JBT1857 <1 3.0  3.7/21.9  6.9/13.6 69 51

Affinity of the peptides to human TFPI measured via BiaCore was lessthan 1 nM. Affinity measured by ELISA (IC₅₀) was 4.8 nM for JBT1837 and2.5 nM for JBT1857. JBT1837 dissociated from human TFPI more slowly thanJBT1857 (i.e., JBT1837 remained bound to human TFPI for a longer periodof time compared to JBT1857). A FXa inhibition assay was performed usingboth full length human TFPI (“flTFPI”) and truncated human TFPI (254amino acids “R&D TFPI”) (0.1 nM FXa, 0.5 nM TFPI, 0.25% DMSO). Activityof the truncated TFPI was fully inhibited by both JBT1837 and JBT1857 at0.5 nM TFPI, while full length TFPI was inhibited 85% and 95% by JBT1857and JBT1837, respectively. At higher concentrations of flTFPI (e.g., 10nM flTFPI), JBT1837 fully inhibited TFPI activity, while JBT1857partially inhibited TFPI activity. EC₅₀'s also were higher when flTFPIwas used in the FXa inhibition study.

In the extrinsic tenase assay, about 85% of truncated TFPI was inhibitedby both peptides. Full length TFPI activity was inhibited about 56% and48% by JBT1837 and JBT1857, respectively. Surprisingly, in thecell-based extrinsic tenase assay, JBT1837 inhibited the activitycell-associated TFPI by about 50% whereas JBT1857 almost fully inhibitedcell-bound TFPI activity. In the plasma-based functional assay, JBT1837inhibited TFPI more efficiently than JBT1857 in human FVIII-inhibitedplasma and FIX-deficient plasma. JBT1837 corrected blood coagulationparameters in FVIII-inhibited blood in the ROTEM assay described inExample 8. JBT1857 also positively impacted blood coagulationparameters, but performed less efficiently than JBT1837 in the assay.

This example compared the affinity and TFPI-inhibitory activity ofcyclic and linear TFPI-binding peptides that target different regions ofthe TFPI protein. JBT1837 (a cyclic peptide belonging to family JBT0120)and JBT1857 (a linear peptide belonging to family JBT0047) efficientlybind human TFPI with affinities less than 1 nM and are potentinhibitors. FXa-TFPI interaction is fully blocked at low TFPIconcentrations by both peptides, while TFPI inhibition by JBT1857 isreduced in the presence of higher concentrations of TFPI. Both peptidespartially inhibit the activity of full-length TFPI in the extrinsictenase assay, and JBT1857 inhibits TFPI activity to a greater degree inthe cell-based extrinsic tenase assay compared to JBT1837. Compared toJBT1857, JBT1837 more efficiently inhibits TFPI in FVIII-deficientplasma. Both peptides improve coagulation parameters of FVIII-inhibitedhuman whole blood by reducing clot time, while JBT1857 improves clotformation velocity to a lesser degree compared to JBT1837.

Example 10

This example illustrates the in vivo activity of TFPI-binding peptidesof the invention in a clinically-relevant animal model. As describedbelow, an exemplary TFPI-binding peptide significantly reduced bloodloss in an animal when administered with suboptimal doses of FVIII andFIX.

JBT2329, a PEGylated (40 kD) TFPI-binding peptide (JBT0047 family) thatcross-reacts with human and murine TFPI, was tested in tail-tip bleedingmodel in FVIII knock-out mice and FIX knock-out mice. FVIII knock-outmice closely minor the condition of hemophilia A patients, and thetail-tip bleeding model is widely used in research to assess efficacy ofdrugs by measuring, e.g., bleeding time, blood loss or survival. ADVATE,a commercially available rFVIII, served as a reference, and ADVATEbuffer-treated animals served as negative controls. Each group contained16 FVIII knock-out mice (8 female+8 male). JBT2329 (1 mg/kg or 0.1mg/kg) or anti-TFPI antibody (maTFPI; 18 mg/kg) was administered 30minutes before the tail-tip was cut. ADVATE (10 IU/kg or 50 IU mg/kg) orADVATE buffer was administered five minutes before the tail was cut off.Test and control substances were administered as an intravenous bolusvia a lateral tail vein injection. Animals were anaesthetized by anintraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine.Approximately 10 minutes later, 2 mm of the tail-tip was cut off. Thetail-tips were placed in warm saline (approximately 37° C.) and bloodwas collected over an observation period of 60 minutes. The amount ofblood was determined gravimetrically. At the end of the observationperiod of 60 minutes the animals were humanely killed by cervicaldislocation before recovery from anesthesia.

Median total blood loss in buffer-treated animals was 930 mg. Mediantotal blood loss in subjects treated with murine anti-TFPI antibody(maTFPI) was 724 mg. The reduction in median total blood loss was morepronounced when the subjects were administered maTFPI with ADVATE. Acombination of maTFPI+10 IU/kg ADVATE led to a median total blood lossof 136 mg, animals treated with maTFPI+50 IU/kg ADVATE experienced amedian total blood loss of 13 mg. Median blood losses of animals treatedwith either 10 or 50 IU/kg ADVATE alone experienced median blood loss of798 and 364 mg, respectively. The superiority of the combinationtreatment of maTFPI+ADVATE over ADVATE alone was statistically shown formaTFPI+50 IU/kg ADVATE versus 50 IU/kg ADVATE (p=0.0010). Although notstatistically significantly superior, blood loss in animals treated withmaTFPI+10 IU/kg ADVATE was distinctively lower than in animals treatedwith 10 IU/kg ADVATE alone.

Efficacy, defined as statistically significant superiority over bufferat a 2.5% level, was shown for JBT2329 dosed at 1 mg/kg in combinationwith 10 and 50 IU/kg ADVATE and dosed at 0.1 mg/kg in combination with50 IU/kg ADVATE (p<0.0004). Animals treated with JBT2329 in combinationwith ADVATE showed a clinically-relevant reduction in blood loss,although the results were not statistically significant (p≧0.0506).Administration of 1 mg/kg JBT2329 without ADVATE did not reduce mediantotal blood loss over that observed in buffer-treated animals (930 mg).

JBT2329 also was tested in a FIX knock-out tail-tip bleed mouse model,which is a clinically-relevant model for hemophilia B human patients.The methodology was substantially similar to that described above withrespect to the FVIII knock-out model. Instead of ADVATE, a recombinantFIX (rFIX) served as a reference. Median total blood loss inbuffer-treated animals was 935 mg. Median total blood loss in animalstreated with a murine anti-TFPI antibody (maTFPI) was 774 mg. Mediantotal blood loss was reduced further when the animals received combinedtreatment of maTFPI and rFIX. A combination of maTFPI+10 IU/kg rFIX ledto a median total blood loss of 25 mg, while animals treated withmaTFPI+50 IU/kg rFIX exhibited a median total blood loss of 10 mg.Median blood loss of animals treated with either 10 or 25 IU/kg rFIXalone experienced a median blood loss of 888 and 774 mg, respectively.

Efficacy, defined as statistically significant superiority over bufferat a 2.5% level, was shown for JBT2329 when dosed at 1 mg/kg incombination with 10 IU/kg rFIX and at 0.1 mg/kg in combination with 10IU/kg rFIX. The superiority of JBT2329 in combination with rFIX overadministration of rFIX alone was observed (p<0.0172), while treatmentwith 1 mg/kg JBT2329 alone did not lead to a significant reduction inmedian total blood loss compared with buffer-treated animals (p=0.321).

In summary, JBT2329 promoted a clinically-relevant reduction of bloodloss when co-administered with suboptimal doses of FVIII and rFIX at alldoses tested. Furthermore, intravenous administration of JBT2329 waswell tolerated in all subjects across all treatment groups without anysigns of acute toxicity.

Example 11

The TFPI-binding peptides described herein are suitable for detectingTFPI in a sample, such as biological sample. This example describes amethod for detecting TFPI using the inventive peptides in an ELISA-likeassay format.

The peptide sequence of JBT1857 was N-terminally modified by theaddition of a biotinyl-Ttds moiety to generate JBT2271(Biotinyl-Ttds-FQSKpNVHVDGYFERL-Aib-AKL-NH2 (SEQ ID NO: 4033)). A96-well microtiter plate (Maxisorp, Nunc) was coated with 50 μl per wellcoating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.3) containing a rangeof TFPI concentrations (0-3 μg/ml, human recombinant TFPI, R&D Systems)for 1 hour at room temperature. The plate was washed three times with350 μl/well wash buffer (175 mM NaCl, 5 mM CaCl₂, 25 mM HEPES, 0.1%Tween 80, pH 7.35). The plate was then blocked with 100 μl blockingbuffer (2% yeast extract, 175 mM NaCl, 5 mM CaCl₂, 25 mM HEPES, 0.1%Tween 80, pH 7.35) for 1 hour at room temperature. The plate was thenwashed three times with 350 μl wash buffer. Fifty μl of differentlyconcentrated JBT2271 solutions in wash buffer (100-0 nM) were added toeach well. The plate was incubated for 1 hour and washed three timeswith 350 μl wash buffer. To each well, 50 μl streptavidin-horseradishperoxidase conjugate (R&D Systems, 1:200 in wash buffer) is added. Afteran incubation period of 1 hour at room temperature, the plate was washedthree times with wash buffer. Fifty μl TMB solution (SeramunBlau fast,Seramun) was added to each well. After a 1.5 minute incubation at roomtemperature, the reaction was stopped by adding 50 μl 1 M H₂SO₄ perwell. Absorbance was measured in a photometer (Molecular DevicesSpectramax M5) at 450 and 620 nm.

JBT2271 allowed detection of as little as 4.1×10⁻¹⁴ mole of TFPI perwell. The results of the assay described above illustrate that theinventive peptides are powerful tools for identifying and/or quantifyingTFPI in a sample.

Example 12

This example describes conditions for an exemplary k_(off) assay forcharacterizing TFPI-binding peptides.

Wells of a microtiter plate (96 wells, Maxisorp, Nunc) are coated with1.6 nM TFPI in coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.3) fortwo hours at room temperature. The plate is then washed three times with350 μl wash buffer (175 mM NaCl, 5 mM CaCl₂, 25 mM HEPES, 0.1% Tween 80,pH 7.35), and wells are blocked with 100 μl blocking buffer (2% yeastextract, 175 mM NaCl, 5 mM CaCl₂, 25 mM HEPES, 0.1% Tween 80, pH 7.35).If an incubation period of 24 hours is employed, the wells are blockedfor at least one hour. Control wells used for a 15 minute incubationperiod are blocked for an additional 23.5 hours.

For a 24 hour incubation period, the wells are washed three times with350 μl wash buffer and are incubated with 50 μl test peptide in washbuffer. The concentration of test peptide depends on the individual IC₉₀concentration determined in, e.g., the TFPI IC₅₀ ELISA assay describedherein. The TFPI-coated wells are exposed to test peptide forapproximately 15 minutes. The wells are subsequently washed three timeswith 350 μl wash buffer and 50 μl tracer peptide (competitor) is added.An exemplary tracer peptide is JBT2271 (1.13 nM in wash buffer). Controlwells (maximum signal) are incubated with tracer only. Blank wellslacking TFPI are incubated with tracer only. Addition of the tracerpeptide commences the 24 hour incubation period.

A 15 minute incubation period is employed as a control if the IC₉₀concentration of the test peptide leads to a 90% reduction of themaximum signal. Wells blocked for an additional 23.5 hours are washedthree times with 350 μl wash buffer to remove the blocking buffer.Subsequently, 50 μl analyte in wash buffer is added and the wells areincubated for 15 min. The concentration of test peptide utilized dependson the peptide's IC₉₀ concentration determined using, e.g., a TFPI IC₅₀ELISA assay. The 15 minute incubation is followed by three washes with350 μl wash buffer and addition of 50 μl tracer peptide. Control wells(maximum signal) are incubated with tracer only. Blank wells lackingTFPI also are incubated with tracer only.

The plate is washed three times with 350 μl wash buffer, and 50 μlstreptavidin-horseradish peroxidase conjugate (R&D Systems, 1:200 inwash buffer) is added to each well. After an incubation period of onehour at room temperature, the plate is washed three times with washbuffer. TMB solution (50 μl per well; SeramunBlau fast, Seramun) isadded. After a 1.5 minute incubation at room temperature, the reactionis stopped by the addition of 50 μl 1 M H₂SO₄ per well. Absorbance ismeasured using a photometer (Spectramax M5, Molecular Devices) at 450and 620 nm. The assay results are presented as a percentage of thecorrected optical density (OD450-OD620) of wells exposed to test peptideand tracer peptide in relation to TFPI-coated wells exposed only totracer.

Example 13

TFPI inhibits FVIIa/TF activity by binding to FVIIa via Kunitz domain 1(KD1). This example describes an exemplary method for evaluating theinfluence of TFPI-binding peptides on TFPI's inhibition of FVIIa/TF.

Kinetic measurements were performed in 25 mM HEPES, 175 mM NaCl, 5 mMCaCl₂, 0.1% BSA, pH 7.3 at 25° C. in 96-well microtiter plates. Twentyμl soluble tissue factor (residues 33-251; Creative Biomart) and 20 μlFVIIa (ERL) at final concentrations of 100 nM and 5 nM, respectively,were mixed and incubated for 15 minutes. Twenty μl of TFPI-bindingpeptide in varying final concentrations (0-2 μM) were added to themixture and incubated for a further 15 minutes. In order to measure theresidual activity of the FVIIa/sTF complex, the reaction mixture wasincubated for 60 minutes with 20 μl TFPI (200 nM). The reaction wasinitiated by the addition of a chromogenic substrate, Chromozym-tPA(Roche) (1 mM). The change in absorbance at 405 nm was monitored byusing a Labsystems iEMS ELISA Reader for 30 minutes. FVIIa/sTF activitymeasured in the absence of TFPI was considered “100% activity” in thecontext of the assay. By plotting peptide concentration against residualactivity, EC₅₀ values were determined.

JBT1857 and JBT1837 were screened against TFPI160, TFPI1-150-Thrombin,NTermKD1, KD1, and KD2 (negative control). JBT1857 demonstrated an EC₅₀of approximately 0.21-0.23 μM for TFPI160, TFPI1-150-Thrombin, NTermKD1,and KD1. JBT1837, which binds KD1 and KD2, demonstrated an EC₅₀ ofapproximately 0.17-0.19 μM for TFPI160 and TFPI1-150-Thrombin, whileactivity in assays involving NTermKD1 and KD1 was approximatelybackground.

The results described above demonstrate that TFPI-binding peptidesefficiently inhibit TFPI-FVIIa/TF interaction. JBT1857 efficientlyinhibited TFPI fragments containing KD1 as a minimal functional entity.Thus, this enzymatic assay confirms X-ray crystallographic data placingthe binding site of JBT1857 within KD1. JBT1837 inhibited TFPI fragmentscontaining the first two Kunitz domains, suggesting that the JBT1837binding site(s) are located within KD1-linker-KD2 region of TFPI. Acombination of Kunitz domains and fragments of a thrombin cleaved TFPI(1-150) did not restore inhibitory activity of JBT1837 in thechromogenic assay. The enzymatic assay described herein is a suitablesurrogate for detecting binding of a TFPI-binding peptide (or a testcompound) to TFPI, and is useful for examining the TFPI-inhibitoryeffect of TFPI-binding compounds.

Example 14

This example describes the influence of PEG and HSA conjugation onexemplary TFPI-binding peptides in vivo.

For pharmacokinetic analysis, C57B16 mice were treated with variousTFPI-binding peptides conjugated to different molecular weight PEGs andHSA. The dose of the peptide-PEG and peptide-HSA conjugates wasnormalized to 1 mg/kg (peptide content). Normalization assurescomparability between the conjugates of different molecular weight. Thepeptide conjugates were dissolved in 175 mM NaCl, 25 mM HEPES pH 7.35and administered intravenously via the tail vein or subcutaneously inthe neck region. Blood draws were taken from three animals (retrobulbar) and collected in heparinized vials at several time pointsfollowing administration. The samples were centrifuged, and thepeptide-conjugate content in plasma was quantified by ELISA.

FIG. 63 illustrates the concentration of PEGylated TFPI-peptidesdetected in plasma at several time points following administration, andTable 17 provides detailed information about the terminal half life andbioavailability of JBT2325-JBT2329, JBT2401, JBT2404 and JBT2410.

TABLE 17 JBT2325 JBT2326 JBT2327 JBT2328 JBT2329 JBT2401 JBT2404 JBT2410HL_λ_z [h] 0.16 0.35 4.2 10.1 19.8 20.7 12.3 7.8 (intravenous)Bioavailability [%] 58.2 76.0 89.7 52.0 73.3 58.4 59.3 46.6 (s.c.)

JBT2329, JBT2401 and JBT2404 are peptides conjugated to 40 kDa linearPEG (JBT2329 and JBT2404) or 40 kDa branched PEG (JBT2401). The 40 kDaconjugates exhibited a longer terminal half-life (HL_λ_z) compared topeptides conjugated to smaller PEGs following intravenousadministration. The area under curve (AUC) of the concentration-timecurve resulting from subcutaneous administration of the peptides wascompared to the AUC generated following intravenous administration tocalculate the bioavailability of the peptides. Results are shown inTable 16. The data demonstrate that TFPI-binding peptide conjugation tohigher molecular weight molecules allows a subcutaneous bioavailabilityof more than 30%.

FIGS. 64A-C illustrate the pharmacokinetic profile of JBT2401, JBT2404,and JBT2410 resulting from subcutaneous and intravenous administrationof the peptides to mice. JBT2404 comprises a PEG conjugated to cysteinein position X4014 relative to formula (XI). JBT2401 comprises a branchedPEG, and JBT2410 is conjugated to HSA. FIG. 64A demonstrates that fusionof a higher molecular weight molecule to a TFPI-binding peptide at aninternal position increases half life. Half life also is increased ifusing branched PEG (JBT2401) and HSA, which increased the in vivo halflife of JBT2410 compared to conjugates having smaller-sized PEGs (e.g.,JBT2325) or free peptide (see FIG. 31).

This example illustrates that the in vivo properties of various peptidesdescribed herein can be improved by conjugation with higher molecularweight molecules (like PEG) and/or with nFcR ligands (like HSA).

Example 15

The following example describes the characterization of JBT1837'sinteraction with KD1-KD2 of TFPI, alone and in the presence of JBT1857,via X-ray crystallography.

Native-PAGE

Native-PAGE was performed to confirm that TFPI-binding peptides thatbind different regions of TFPI could bind TFPI KD1-KD2 simultaneously.Fifteen percent polyacrylamide-gels (without SDS) were used for thenative gel-electrophoresis on samples comprising (1) a TFPI fragmentcomprising KD1-KD2, (2) KD1-KD2 and JBT1857, (3) KD1-KD2 and JBT1837,and (4) KD1-KD2 with both JBT1857 and JBT1837. Glycerine and Ponceau redwere added to the protein samples. The anode buffer was obtained bydissolving 25 mM imidazole in 1 liter water and adjusting the pH to 7with HCl. Clear cathode buffer was prepared by dissolving 7.5 mMimidazole and 50 mM tricine in one liter of water. Water was added toeach solution to reach a final volume of two liters. Serva Blue G (0.02%w/v final) was added to two liters of cathode buffer to generate bluestock 10× cathode buffer (blue stock 10×). Gels were run for 30 minutesat 80 V, then for about three hours at 250 V. The gels were stained andde-stained as described in Chakavarti et al., J Vis Exp., 12; (16)(2008).

KD1-KD2 was visualized in the gel as a single band. Samples comprisingK1-K2 in combination JBT1857 and/or JBT1837 resulted in a shift of theinitial KD1-KD2 band to a higher molecular weight to charge ratio. Aftermixing of KD1-KD2 and JBT1837, partial precipitation was observed,indicating a decrease in solubility after complex formation. JBT1857shifted the complete KD1-KD2 band, while JBT1837 shifted approximately ⅓of the initial band. Additionally, in the presence of both JBT1837 andJBT1857, the KD1-KD2 band shifts to the highest molecular weight tocharge ratio, indicating the formation of a ternary complex consistingof KD1-KD2, JBT1837 and JBT1857. Thus, JBT1837 and JBT1857 can bind TFPIsimultaneously.

Materials and Methods—Interaction of JBT1837 with KD1-KD2

Crystal Growth:

Initial crystals were formed at 20° C. by mixing 0.4 μl of a 1:1 complex(1.5 mg/ml; 10 mM Tris/8.0, 100 mM NaCl) with 0.2 μl precipitant (20%w/v PEG 6000, 50 mM Imidazole pH 8.0). Crystals were reproduced at 20°C. by mixing 1 μl protein with 0.5 μl precipitant (20% w/v PEG 6000, 50mM Imidazole pH 8.0). A cryoprotectant (30% w/v PEG 6000, 34% glycerol)was added to the crystal drop (final concentration of 25% glycerol) toprotect resulting crystals from disorder and ice formation during flashcooling with liquid nitrogen.

Data Processing:

Diffraction data were collected from 87.26 Å to 1.95 Å and evaluatedusing the MOSFLM and iMOSFLM packages (Leslie, (2006) Acta Crystallogr DBiol Crystallogr 62, 48-57), followed by merging and scaling using CCP4suite (McCoy et al., (2007) J Appl Crystallogr 40, 658-674). Crystalsbelong to the orthorhombic space group P 2₁2₁2₁ with unit celldimensions of a=42.35 Å, b=44.13 Å, c=174.53 Å, α=β=γ=90.0° and 2complexes in the asymmetric unit. The Patterson search was carried outusing the PHASER program (McCoy, supra) and two search ensembles, acrystal structure of KD2 (1TFX) (Burgering et al. (1997) J Mol Biol 269,395-407) and a previously solved KD1 crystal structure. The unit cellcontained approximately 54% solvent. Non-crystallographic electrondensity averaging, model building and model refinement was carried outwith the programs Coot and Refmac (McCoy, supra; Murshudov et al, (1997)Acta Crystallogr D Biol Crystallogr 53, 240-55). The current model iscompletely defined for both copies of the JBT1837 peptide (23AA) and theinteraction with the protein with current R=0.213, R_(free)=0.245,deviation from ideal geometry rms(bond)=0.0098 Å, rms(angle)=1.28°.

Analysis of the Interaction Surface:

The interaction surface of KD1-KD2 and JBT1837 was analyzed withPDBePISA. The impact of single amino acids was weighted by the totalreduction of their solvent accessible area after complex formation(buried solvent accessible area BSAA) and considered by generating a2D-interaction-matrix.

Materials and Methods—Interaction of KD1-KD2 with JBT1837 and JBT1857

Crystal Growth:

Initial crystals where formed at 20° C. by mixing 0.4 μl of a 1:1:1complex (1.5 mg/ml; 10 mM Tris/8.0, 100 mM NaCl) with 0.2 μl precipitant(2M (NH₄)₂SO₄, 5% PEG 400, 100 mM MES pH 6.5). Crystals were reproducedand optimized at 20° C. by mixing 1 μl protein with 0.5 μl precipitant(1.5 M (NH₄)₂SO₄, 5% PEG 400, 100 mM MES pH 6.5). A cryoprotectant (2M(NH₄)₂SO₄, 25% glycerol) was added stepwise to the crystal drop (finalconcentration of 2.3M (NH₄)₂SO₄ and 20% glycerol) to protect theresulting crystals from disorders and ice formation during flash coolingwith liquid nitrogen. The drop was covered with cryo-oil and crystalswere harvested.

Data Processing:

Diffraction data were collected from 74.3 Å to 2.7 Å and evaluated usingthe MOSFLM and iMOSFLM packages, followed by merging and scaling usingCCP4 suite (Leslie, (2006) Acta Crystallogr D Biol Crystallogr 62,48-57; Acta Crystallogr D Biol Crystallogr 50, 760-3 (1994)). Crystalsbelong to the tetragonal space group P 4₃2₁2 with unit cell dimensionsof a=79.07 Å, b=79.07 Å, c=218.17 Å, α=β=γ=90.0°.

X-Ray Crystallography Results

The KD1-KD2 structure is defined in the electron density from L21through E148; while chemically present, residues D149 and G150 areconformationally disordered in two crystallographically-independentcopies of the molecule.

Both domains show a Kunitz-type structure. Only ˜⅓ of the structure isengaged in secondary structure elements; these are two short α-helicalelements at S24-A27(KD1)/D95-F98(KD2) (α1/α3) and L69-M75/L140-E148(α2/α4) and a two-stranded β sheet comprising M39-N45/I110-N116(131/(33) and R49-155/K120-K126 (β2/β4). These elements form thetopological framework that is stabilized by the three canonicaldisulfide bonds involving C26-C76, C35-059, and C51-C72 in KD1 andC95-C147, C106-C130, and C122-C143 in KD2.

This is the first structure of TFPI consisting of KD1, KD2 and theirlinker elucidated by X-ray crystallography. Remarkably, JBT1837 locksKD1-KD2 in a distinct conformational state in which both Kunitz-domainsare related via a two-fold symmetry. Additionally the conformation ofTFPI is intrinsically stabilized by two turns, a β-turn (tβ) fromT77-A81 and a γ-turn (tγ) from Q90-K93. tβ is stabilized by threehydrogen bonds (O K74-N N82, O N80-Nζ K74, O N82-Nε H23) and leads to ashortening of α2 in KD1 by two residues compared to homologKunitz-domains and the crystal structure of KD1 alone. tγ is stabilizedby four hydrogen bonds (O T88-N D95, O Q90-N K93, O K93-N Q90, Oγ T88-OδD95).

The 23 mer peptide JBT1837 assumes a β-hairpin like structure which canbe segmented into (i) the pin, a two-stranded β sheet comprisingY2_(AP)-A8_(AP) and T17_(AP)-F23_(AP); (ii) and the needle eye, a β-turncomprising D11_(AP)-T15_(AP). (The subscript _(AP) indicates thesequence numbering in the antagonistic peptide (JBT1837).) The β-sheetis stabilized by a disulfide bridge (C7_(AP) and C18_(AP)) and ahydrophobic zipper comprising the side chains of Y3_(AP), W5_(AP) andW20_(AP).

Analyzing the interaction between KD1-KD2 and JBT1837 with the PISAserver resulted in a total interaction surface of 1340 Å². More than ⅔of the interaction surface consists of a hydrophobic anchor in JBT1837interacting with residues spread over TFPI, including KD1, KD2, and thelinker, as illustrated by an interaction matrix, a summary of which isprovided in Table 18, and FIG. 74.

TABLE 18 JBT BSAA 1837 {acute over (Å)}² % KD1 LINKER KD2 Ser1 50.7 35C59, E60 Tyr2 83.9 72 R41, Y56 Tyr3 11.7 12 R65 Lys4 48.0 72 E67 E142Trp5 143.5 96 Q63, M75 I84 His6 83.9 89 M134, F137, E142, I146 Cys7 26.865 E71 Ala8 36.9 89 M134, N136 Met9 95.0 63 R83 L131, G132, N133, M134Arg10 52.5 31 L131 Asp11 41.2 61 R83 L131 Met12 55.2 28 C106, C130 Lys1334.7 19 N80 Gly14 17.2 57 Thr15 75.7 94 K74 N80, N82, R83 Met16 33.26 42N82, R83 Thr17 71.5 89 R83, I85 M134 Cys18 60.9 100 M75 N82, R83, I84,I85 Val19 74.65 99 I85, T87 F96, M134 Trp20 89.4 68 Q63, R65 T87 Val 2154.25 99 T87 I146 Lys22 37.1 27 Phe23 108.5 64 N145, I146

In addition to the hydrophobic contacts, the matrix identifies polarinteractions that stabilize the TFPI-JBT1837 complex, e.g., by hydrogenbonds between the side chain of H6_(AP) with the carbonyl oxygen of M134and NE of W5_(AP) with the carbonyl oxygen of Q63, as well as a shortβ-strand comprising M16_(AP)-C18_(AP) and R83-I85. Additionally Nζ ofK4_(AP) is equidistantly coordinated by the side chains of E67 and E142.

Beside its role in JBT1837 stabilization, the disulfide bridge ofC7_(AP)-C18_(AP) perfectly fits into a hydrophobic cavity formed by E71,M75, N82, and 184, thus playing a significant role in stabilizing theTFPI-JBT1837 complex.

Although TFPI is mainly conserved through different species, the keyresidues within the linker and KD2 (M134, I146) are only shared by closerelatives to the human species (FIG. 74). Additionally, the A81Vsubstitution in macaque destabilizes the β-turn of the linker by sterichindrance, impairing subsequent interaction of the linker with JBT1837.

KD1 of the previously solved KD1+JBT1857 complex was superimposed on theKD1-KD2+JBT1837 complex to provide a model of the arrangement of theternary structure. The model suggested that JBT1837 and JBT1857 bind toopposing sites of KD1. The C-terminus of JBT1857 and the N-terminus ofJBT1837 were 20 Å apart; thus, a linker moiety approximately 20 Å inlength, corresponding to about 10 amino acids, would connect JBT1857 andJBT1837 and allow binding to the subunits respective binding sites onTFPI.

KD1-KD2+JBT1837+JBT1857 crystals belong to the tetragonal space group P4₃2₁2 and diffract to a maximum resolution of 2.7 Å. Screw axes wereconfirmed by axial extinctions and cell content analysis indicated amass of 72 kDa in the asymmetric unit. Assuming that the ternary complexcrystallized, this corresponds to either three (62 kDa; 61% solvens) orfour (82 kDa; 46% solvens) copies in the asymmetric unit. However, thestructure could not be completely solved, possibly due to the inherentflexibility of the KD1-KD2 molecule structure. Analysis of theinteractions between TFPI and JBT1837, as well as the peptide-inducedconformation of TFPI, suggests that JBT1837 may not bind to TFPI ofspecies other than human. The high selectivity of JBT1837 isadvantageous as it minimizes cross-reactivity and thus unwanted sideeffects. JBT1857 binds TFPI at a different site, and native PAGEdemonstrated that both peptides can bind to TFPI at the same time. Thisanalysis is further confirmed by a crystal structure-based model ofKD1-KD2+JBT1837+JBT1857 (FIG. 75). The C-terminus of JBT1857 and theN-terminus of JBT1837 lie only 20 Å apart, and a linker of e.g., ten Ala(or Ser) would allow binding of both JBT1837 and JBT1857 to theirbinding sites within TFPI.

Example 16

The following example describes the characterization of a peptidecomplex that binds TFPI.

TFPI-binding peptides were produced as described herein. The peptideswere synthesized as trifluoroacetate (TFA) salts with a purity >90%, andsolved in DMSO to a stock concentration of 10 mM.

Competition (IC₅₀) ELISAs were performed using biotinylated TFPI-bindingpeptides as “tracers” to compete for TFPI-binding with non-biotinylatedcandidate peptides. The assay principle is depicted in FIG. 6B.Ninety-six well Maxisorp plates (Nunc) were coated with 0.05 μg/mL TFPIin coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) overnight. Plateswere washed three times with 350 μl wash buffer (HNaT: 175 mM NaCl, 25mM HEPES, 5 mM CaCl₂, 0.1% Tween 80, pH 7.35), and blocked with 200 μl2% yeast extract in HNaT for two hours. Plates were then washed threetimes with 350 μl HNaT. Biotinylated tracer peptides were applied at aconcentration corresponding to their respective EC₉₀ values determinedin a binding ELISA (mean value if n>2). A competitor stock solution ofcandidate peptide (10 mM or prediluted in DMSO) was diluted 1/33.3 inHNaT, and a serial 1/3 dilution was prepared with HNaT with 3% DMSO. Thedilution strategy employed in a particular assay was adjusted based onthe affinity of the peptide. The dilution was further diluted with thebiotinylated tracer peptide in a ratio of 1:6 (20 μl competitor dilutionand 100 μl tracer peptide). The mixture of competitor and tracer peptidewas applied to the TFPI-coated microtiter plate and incubated for 1.5hours. The plates were washed three times with 350 μl HNaT. Peptide-TFPIbinding was detected by applying horseradish peroxidase (HRP)-conjugatedstreptavidin to the microtiter plate, incubating the mixture for onehour, washing the plate three times with 350 μl HNaT, applying TMB(3,3′5,5′-Tetramethylbenzidin), and detecting the subsequent chromogenicconversion of TMB by HRP. IC₅₀ graphs for the representative peptidesJBT1857 (SEQ ID NO: 178), JBT1837 (SEQ ID NO: 1044) and the peptidecomplex JBT2547(Ac-FQSKpNVHVDGYFERL-Aib-AKLSSSSSSSSSSSYYKWH[CAMRDMKGTMTC]VWVKF-NH2 (SEQID NO: 4260)) are depicted in FIGS. 66A and 66B.

The TFPI-inhibitory peptide complex JBT2547, comprising two differentTFPI-binding peptides (JBT1857 and JBT1837) that bind two differentsites within TFPI, demonstrated an IC₅₀ of 1.33×10⁻⁹ M (Tracer JBT2271)and an IC₅₀ of 5.57×10⁻¹° M (tracer JBT2316), which was comparable orlower than the IC₅₀ of the peptide subunits.

Additionally, JBT2547 binding to full length TFPI was characterized in asurface plasmon resonance assay (BIAcore T200™, GE Healthcare, ChalfontSt. Giles, UK). Recombinant, full length TFPI-1 was immobilized on a CMSchip aiming at 500 RU. Peptide was injected at a flow rate of 30μL/minute at concentrations ranging from 1 to 16 nM in HBS-P buffer, pH7.4, 0.1% DMSO. Subsequently, peptide was dissociated for 600 seconds.BIAcore T200™ Evaluation Software was utilized to analyze the data,which revealed that JBT2547 tightly binds full length TFPI with abinding constant <1 nM. The K_(D) was calculated to be 7.49×10⁻¹²M.

Example 17

This example describes an exemplary method for characterizing thebinding affinity of a TFPI-binding peptide complex using a k_(off)assay.

Wells of a microtiter plate (96 wells, Maxisorp, Nunc) were coated with1.6 nM TFPI in coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.3) fortwo hours at room temperature. The plate was then washed three timeswith 350 μl wash buffer (175 mM NaCl, 5 mM CaCl₂, 25 mM HEPES, 0.1%Tween 80, pH 7.35), and the wells were blocked with 100 μl blockingbuffer (2% yeast extract, 175 mM NaCl, 5 mM CaCl₂, 25 mM HEPES, 0.1%Tween 80, pH 7.35). If an incubation period of 24 hours was employed,the wells were blocked for at least one hour. Control wells used for a15 minute incubation period were blocked for additional 23.5 hours.

For a 24 hour incubation period, the wells were washed three times with350 μl wash buffer and incubated with 50 μl test peptide (JBT2547 (SEQID NO: 4260) or JBT1857 (SEQ ID NO: 178)) in wash buffer. Theconcentration of test peptide was adjusted based on the individual IC₉₀concentration determined in, e.g., the TFPI IC₅₀ ELISA assay describedherein. The TFPI-coated wells were exposed to test peptide forapproximately 15 minutes. The wells were subsequently washed three timeswith 350 μl wash buffer, and 50 μl tracer (competitor) peptide (JBT2271(SEQ ID NO: 4033)) was added. Control wells (maximum signal) wereincubated with tracer only. Blank wells lacking TFPI-coating wereincubated with tracer only. Addition of the tracer peptide started the24 hour incubation period.

A 15 minute incubation period was employed as a control if the IC₉₀concentration of the test peptide led to a 90% reduction of the maximumsignal. Wells blocked for an additional 23.5 hours were washed threetimes with 350 μl wash buffer to remove the blocking buffer.Subsequently, 50 μl analyte in wash buffer was added, and the wells wereincubated for 15 minutes. The concentration of test peptide utilized wasadjusted based on the peptide's IC₉₀ concentration determined using,e.g., a TFPI IC₅₀ ELISA assay. The 15 minute incubation was followed bythree wash steps with 350 μl wash buffer and the addition of 50 μltracer peptide. Control wells (maximum signal) were incubated withtracer only. Blank wells lacking TFPI also were incubated with traceronly.

The plate was washed three times with 350 μl wash buffer, and 50 μlHRP-conjugated streptavidin (R&D Systems, 1:200 in wash buffer) wasadded to each well. After an incubation period of one hour at roomtemperature, the plate was washed three times with wash buffer. TMBsolution (50 μl per well; SeramunBlau fast, Seramun) was added. After a1.5 minute incubation at room temperature, the reaction was stopped bythe addition of 50 μl 1 M H₂SO₄ per well. Absorbance was measured usinga photometer (Spectramax M5, Molecular Devices) at 450 nm and 620 nm.

The assay results are presented in FIG. 67 as a percentage of thecorrected optical density (OD450-OD620) of wells exposed to test peptideand tracer peptide in relation to TFPI-coated wells exposed only totracer. After 24 hours, the TFPI-binding peptide complex continued toblock binding of the tracer to TFPI. In contrast, JBT1857 dissociatedfrom TFPI during the 24 hour incubation period. Thus, JBT2547dissociated significantly slower from TFPI than one of its peptidesubunit, JBT1857.

The affinity of JBT2528 for TFPI also was determined using the methodsdescribed herein: K_(D)=1.6 nM; k_(on)=6.5×10⁵ 1/Ms; k_(off)=9.7×10⁴1/s. JBT2528 binds TFPI KD1.

Example 18

This example describes the plasma stability of the TFPI-binding peptidecomplex JBT-2547 in mouse and human plasma. JBT2547 (SEQ ID NO: 4260)and JBT1857 (SEQ ID NO: 178) were added to samples of mouse or humanplasma and incubated for 24 hour at 37° C. The percentage of the initialamount of peptide remaining after incubation was determined by IC₅₀ELISA on Maxisorp plates coated with 0.05 mg/ml TFPI (2.26 nM tracerpeptide JBT2271). Less than 5% of the initial amount of JBT1857 remainedin mouse plasma after the incubation period, while 15% of the initialamount of JBT-2547 remained after the same incubation period. Similarly,JBT2547 was more stable in human plasma compared to JBT1857 and JBT1837;27% of the original amount of JBT1857 and 46% of JBT1837 remained after24 hours while 54% of the original amount of JBT2547 was detected. Thus,the complex of JBT1857 and JBT1837 increased stability and resistance toplasma proteases. JBT2528 (Hex-FQSKp-C(Acm)-VH-Tle-DaYFERL-Aib-AKL-NH2(SEQ ID NO: 4246)) also exhibited enhanced stability: 93% of theoriginal amount of remained after 24 hours in human plasma, and 35% ofthe original amount of remained after 24 hours in mouse plasma.

Example 19

The following example describes the characterization of theTFPI-inhibitory activity of a TFPI-binding peptide complex andTFPI-binding peptide monomers using FXa inhibition and extrinsic tenaseinhibition assays described in Example 3.

Peptides were diluted in 1.25× reaction buffer+0.1% Tween-80 (31.25 mMHEPES; 218.75 mM NaCl; 6.25 mM CaCl₂; 0.125% BSA; pH 7.35) from 1 or 10mM stocks (in DMSO). TFPI, FVIIa, and lipidated TF were diluted in 1.25×reaction buffer. Phospholipid vesicles (DOPC/POPS 80/20) and chromogenicsubstrate specific for FXa (S-2222 (available from DiaPharma, WestChester, Ohio)), all diluted in Aqua dest., were added to 96-wellplates. After an incubation period, TFPI and peptide dilutions wereadded. The TFPI concentration in the extrinsic tenase inhibition assaywas 0.0625 nM. FX activation was initiated by adding FX to the wells.FXa-mediated chromogenic substrate conversion was determined byobserving an increase in absorbance using a micro-plate reader. Theamount of FXa generated at certain time points was calculated from theOD readings. FXa generated at 20 minutes after start of the reaction wasconsidered for calculation of EC₅₀ from plots of peptide concentrationversus the inhibition of TFPI (%).

The functional inhibition of TFPI also was examined using a FXainhibition assay. A FXa-specific chromogenic substrate (S-2222) andphospholipid vesicles (DOPC/POPS 80/20), both diluted in Aqua dest., andTFPI proteins (full length human TFPI, human TFPI 1-160, murine TFPI1-160, and cynomolgus TFPI 1-160) diluted in 1.25× reaction buffer, wereadded to 96 well plates. The TFPI concentration in the FXa inhibitionassay was 0.5 nM. Peptides were diluted from 1 or 10 mM stocks (in DMSO)in 1.25× reaction buffer+0.1% Tween-80. The peptide dilutions (2.5 μl)were added to the 96 well plates. The conversion of chromogenicsubstrate was triggered by the addition of FXa, and the kinetics of theconversion was measured in a micro-plate reader. OD readings after 115minutes were considered for calculation of the EC₅₀ from plots ofpeptide concentration versus the inhibition of TFPI.

Results from the FXa inhibition assay and extrinsic tenase assay areprovided in Table 19 and FIGS. 68A and 68B.

TABLE 19 FXa Inhibition Assay Extrinsic Tenase Assay Maximal MaximalEC₅₀ [μM] inhibition (%) EC₅₀ [μM] inhibition (%) JBT1837 1.3 97 0.2 72JBT1857 4.3 90 8.8 59 JBT2528 2.9 89 6.0 71 JBT2547 1.0 98 0.3 99JBT1837 + 0.8 98 0.3 84 JBT1857

JBT1837, JBT2547, JBT2528, and the combination of JBT1837 and JBT1857very efficiently inhibited 0.5 nM full length and C-terminally truncatedTFPI 1-160 (Table 19 and FIGS. 68A and 68B) in the FXa inhibition assay.JBT1857 less efficiently inhibited both TFPIs with EC₅₀s of 4.3 and 3.1nM, respectively. At concentrations above 100 nM, JBT1837, JBT2547, andthe combination of JBT1837 and JBT1857 fully blocked TFPI activity, asindicated by a nearly 100% maximal inhibition. JBT1857 (and JBT2528) isa partial inhibitor of TFPI and demonstrated some residual TFPIinhibitory activity at high and saturating concentrations. Inhibition ofboth full length and C-terminally truncated TFPIs confirms that bindingepitopes of the peptides are within Kunitz Domains 1 and 2, and that theC-terminal region of TFPI is not required for efficient inhibition ofTFPI activity.

The candidate peptides also were analyzed for inhibition of murine andcynomolgus monkey TFPI. C-terminally truncated murine and monkey TFPIproteins (TFPI 1-160) were used in a FXa inhibition assay performed asdescribed herein. The results are summarized in Table 20 and FIGS. 68Cand 68D.

TABLE 20 Cynomolgus Monkey FXa Mouse TFPI 1-160 TFPI 1-160 InhibitionEC₅₀ Maximal EC₅₀ Maximal Assay [μM] inhibition (%) [μM] inhibition (%)JBT1837 — — — — JBT1857 7.2 93 2060*   —* JBT2547 3.1 85 10 100JBT1837 + 7.0 89 100   89 JBT1857 *no maximum reached for fitting

JBT1837 did not inhibit mouse TFPI 1-160 up to 10 μM (FIG. 68C).Cynomolgus monkey TFPI was only weakly inhibited at μM concentrations,likely due to inter-species sequence differences which are incompatiblewith binding of JBT1837 (FIG. 68D). JBT1857 efficiently inhibited mouseTFPI resulting in an EC₅₀ of 7.2 nM, which is comparable to inhibitionof human TFPI. Cynomolgus monkey TFPI was inefficiently inhibited byJBT1857, likely due to an Ala to Pro substitution within the bindingsite of JBT1857.

JBT2547 most efficiently inhibited both mouse and cynomolgus monkeyTFPI. JBT2547 mediated an approximately 200-fold and 10-fold greaterreduction of EC₅₀s compared to JBT1857 and to the combination of JBT1837and JBT1857. This further indicates that the molecular fusion of the twopeptides entities increases its TFPI inhibitory activity.

To demonstrate that the peptide complex efficiently inhibits highconcentrations of TFPI, peptides were titrated at increasing human TFPIconcentrations. JBT2547 stoichiometrically and fully (100%) inhibitedTFPI activity at all concentrations tested (up to 10 nM) (FIGS. 68E and68F). In contrast, peptides JBT1837 and JBT2548 (a derivative ofJBT1857) are partial inhibitors of TFPI at high concentrations (FIG.68D). Linking TFPI-inhibitory peptides to form a peptide complexenhances inhibition of TFPI activity.

In the extrinsic tenase assay, JBT2547 restored extrinsiccomplex-mediated FX activation in the presence of TFPI with an EC₅₀ of0.3 nM, resulting in nearly 100% inhibition of TFPI activity at low TFPIconcentration (0.063 nM). JBT1837, JBT1857 and the combination ofJBT1837+JBT1857 less efficiently inhibited TFPI, as indicated by higherEC₅₀ and reduced maximal inhibition (FIG. 69A). To demonstrate that thepeptide complex efficiently inhibits high concentrations of TFPI,JBT2547 was titrated at increasing TFPI concentrations. JBT2547efficiently inhibited TFPI activity at all concentrations tested (up to10 nM) with lowest EC₅₀s of the peptides tested (FIGS. 69B-69D). Incontrast, peptides JBT1837, JBT2548, and the combination of peptidesJBT1837+JBT1857 partially inhibited TFPI at high concentrations (FIGS.69C-69D) with higher EC₅₀ compared to JBT2547.

TFPI antagonist activities of JBT1837, JBT1857, a mixture of JBT1837 andJBT1857, and JBT2547 also was determined in reactions systems in whichFXa was inhibited by TFPI in the presence of Ca²⁺, phospholipid(PL)+Ca²⁺, and PL+Ca²⁺+protein S. The effectivity by which the peptidesblock the activity of TFPI increases in the order JBT1857, JBT1837, themixture of JBT1837 and JBT1857, and JBT2547. JBT1837 and JBT1857 did notcompletely block TFPI, particularly not in the presence of cofactorprotein S. A mixture of JBT1837 and JBT1857 was more potent as TFPIantagonist than the individual peptides. The fusion peptide JBT2547 wasby far the best TFPI antagonist; at a concentration of 50 nM the peptidecomplex almost completely blocked TFPI even in the presence ofPL+Ca²⁺+protein S.

This example demonstrates that the linkage of two TFPI-binding peptidesof the invention by, e.g., molecular fusion, improves TFPI inhibitoryactivity. JBT1837 prevents the formation of the primary TFPI-FXa complex(encounter complex) and at high concentrations can fully block TFPIinhibition of FXa. JBT1857 prevents the transition of the weak encounterto the tight TFPI-FXa complex and partially inhibits TFPI since in thepresence of JBT1857 it is still possible to form the primary TFPI-FXacomplex (encounter complex). A molecular fusion of the two TFPI bindingpeptides demonstrated a great degree of inhibitory activity than eachpeptide alone and a mixture of the peptide subunits (i.e., the effectwas greater than additive).

Example 20

In this example, the TFPI inhibitory activity of a TFPI-binding peptidecomplex is characterized using the plasma-based assay of Example 4.TFPI-binding peptides were tested under physiological conditions (−0.2nM full length TFPI) as well conditions mimicking elevated full lengthTFPI plasma levels (up to 10 nM full length TFPI).

The influence of peptides on thrombin generation in the absence or inthe presence of exogenous flTFPI was measured in duplicate viacalibrated automated thrombography in a Fluoroskan Ascent® reader(Thermo Labsystems, Helsinki, Finland; filters 390 nm excitation and 460nm emission) following the slow cleavage of a thrombin-specificfluorogenic substrate (Hemker, Pathophysiol. Haemost. Thromb., 33, 4-15(2003)). As a model for antibody-mediated FVIII deficiency, frozenpooled normal plasma (George King Bio-Medical Inc., Overland Park, KN)was incubated with high titer, heat inactivated, anti-human FVIII plasmaraised in goat (4490 BU/ml; Baxter BioScience, Vienna, Austria) givingrise to 50 BU/mL. Assays were also performed with cynomolgus monkey andmarmoset monkey plasma. The plasmas were mixed with corn trypsininhibitor (CTI) (Hematologic Technologies, Inc., Essex Junction, Vt.) toinhibit Factor XIIa contamination, resulting in a final concentration of40 μg/mL.

Pre-warmed (37° C.) plasma (80 μL) was added to each well of a 96 wellmicro-plate (Immulon 2HB, clear U-bottom; Thermo Electron, Waltham,Mass.). To trigger thrombin generation by Tissue Factor, 10 μL of PPPlow reagent containing recombinant human Tissue Factor (12 μM) andphospholipid vesicles composed of phosphatidylserine,phosphatidylcholine and phosphatidylethanolamine (48 μM) (ThrombinoscopeBV, Maastricht, The Netherlands) were added. Just prior to putting theplate into the pre-warmed (37° C.) reader, 5 μL of peptide solutionswere added, resulting in plasma concentrations of 1-100 nM. Finally, 5μL HEPES buffered saline with 5 mg/mL bovine serum albumin(Sigma-Aldrich Corporation, St. Louis, Mo., USA) or 5 μL full lengthTFPI (flTFPI) dilution were added. The flTFPI protein (3557 nM) had beenexpressed in SK Hep cells and purified. Plasma concentrations of flTFPIvaried between 0.31 and 10 nM, which is equivalent to a ˜2 to 50-foldincrease in endogenous flTFPI plasma concentration. Thrombin generationwas initiated by addition of 20 μL of FluCa reagent (Thrombinoscope BV,Maastricht, The Netherlands) containing a fluorogenic substrate andHEPES-buffered CaCl₂ (100 mM). Fluorescence intensity was recorded at37° C. The parameters of the resulting thrombin generation curves werecalculated using Thrombinoscope™ software (Thrombinoscope BV,Maastricht, The Netherlands) and thrombin calibrator to correct forinner filter and substrate consumption effects (Hemker, Pathophysiol.Haemost. Thromb., 33, 4-15 (2003)). The final plasma dilution, TFconcentration, and assay temperature for the human plasma assay was1:1.5, 1 pM, and 37° C., respectively. The final plasma dilution, TFconcentration, and assay temperature for the mouse C57B16 plasma assayand mouse FVII−/− plasma assay was 1:2.4, 0.4 pM, and 33° C.respectively. The final plasma dilution, TF concentration, and assaytemperature for the cynomolgous and marmoset monkey plasma assays was1:1.5, 0.6 pM, and 37° C. respectively.

Representative results for improvement of thrombin generation of humanFVIII inhibited plasma are provided in Table 21.

TABLE 21 Maximal improvement of thrombin generation relative to EC₅₀ aninhibitory polyclonal anti (nM) TFPI antibody (%) JBT1834 4.8 55.9JBT1857 6.8 37.0 JBT1837 + JBT1857 7.4 104.6 JBT2547 7.8 112.7

JBT1837, JBT1857, a mixture of JBT1837+JBT1857, and the complex ofJBT1837 and JBT1857 (JBT2547) improved thrombin generation ofFVIII-inhibited plasma with similar EC₅₀s. The maximal efficacy relativeto a polyclonal anti-TFPI antibody was highest for JBT2547,demonstrating that fusion of two TFPI-binding peptides described hereinenhances inhibition of TFPI activity in plasma.

FIGS. 70A-70C illustrate the inhibitory activity of 1-100 nM of thefusion peptide JBT2547 (diamond) toward elevated levels of flTFPI plasmalevels (1.25, 3.75 and 10 nM) in the thrombin generation assay. Incomparison with JBT1837 (triangle) and JBT1857 (circle), JBT2547 shows asubstantially greater ability to increase the thrombin peak even in thepresence of very high flTFPI plasma levels. Although combining JBT1837and JBT1857 (square) improved the response over each monomeric peptidealone, the combination of monomers did not achieve the level ofactivation achieved by the peptide complex. The TFPI-antagonisticpotential of JBT2547 also was tested in the presence of a wideconcentration range of flTFPI of up to 10 nM, which is equivalent to50-fold higher than physiological flTFPI plasma concentration.Concentrations of 50-100 nM JBT2547 fully compensated the anticoagulanteffect of 10 nM flTFPI, and thrombin peak values reached normal plasmalevels or above. JBT2547 concentrations below 50 nM improved thrombingeneration of FVIII-inhibited plasma in a flTFPI-dependent manner.

The results of thrombin generation experiments with several animalplasmas are summarized in Tables 22 and 23.

TABLE 22 Mouse C57Bl6 plasma Mouse FVIII knock out EC₅₀ Maximal EC₅₀Maximal [nM] inhibition (%) [μM] inhibition (%) JBT1837 No No No Nobinding binding binding binding JBT1857 64.1 115.8 55.1 134.4 JBT254741.0 109.3 56.7 138.3 JBT1837 + 52.7 104.7 61.1 144.0 JBT1857

TABLE 23 EC₅₀ [nM]; EC₅₀ [μM]; Cynomologus Monkey Marmoset PlasmaJBT1837 — No binding JBT1857 1003.7 35.1 JBT2547 — 38.4 JBT1837 + — 34.8JBT1857 * “—” indicates no curve fit

JBT1837 did not inhibit mouse and marmoset monkey TFPI at relevantconcentrations. Cynomolgus monkey TFPI is a poorly inhibited only at μMconcentrations. JBT1857 efficiently inhibited mouse and marmoset monkeyTFPIs, whereas cynomolgus monkey TFPI was less efficiently inhibited byJBT1857, likely due to an Ala to Pro amino acid substitution within thebinding site of JBT1857 which is conserved as an Ala in human, mouse andmarmoset monkey TFPI. Cynomolgus monkey TFPI is most efficientlyinhibited by the fusion peptide JBT2547.

Example 21

This example describes the ability of the TFPI-binding peptidesdescribed above to restore extrinsic tenase complex-mediated conversionof FX to FXa in a cell-based extrinsic tenase assay. The assay wasperformed as described in Example 5. FXa concentrations obtained afternine minutes of reaction in the presence of 100 nM polyclonal anti-humanTFPI antibody (R&D Systems, AF2974) were set to 100%, and the samplesthat were not exposed to antibody were set to 0% TFPI inhibitory effect,allowing for assessment of peptide's inhibitory activity. The results ofthe assay are illustrated in FIGS. 71A and 71B. JBT2547 demonstratedimproved inhibition of HUVEC TFPI compared to JBT1837, JBT1857, JBT2548,and a mixture of JBT1857 and JBT1837. Full inhibition of TFPI wasachieved at concentrations as low as about 10 nM.

Example 22

This example describes a method for assessing clot formation in wholeblood using rotation thromboelastography (ROTEM). The assay wasperformed as described in Example 8. The analytical set-up was asfollows: 300 μL of pre-warmed (37° C.) CTI treated citrated whole bloodwas added to a peptide sample (10, 100, 1000 nM final assayconcentration) or a control, followed by addition of a 1:15 dilution ofTF PRP reagent containing recombinant human tissue factor (rTF, 3 pM)(TS40, Thrombinoscope BV, Maastricht, The Netherlands). In certainexperiments, exogenous full length TFPI (flTFPI) at final concentrationsof 2 or 10 nM was added to simulate flTFPI plasma levels of up to50-fold over normal. Coagulation was initiated by the addition of 20 μL200 mM CaCl₂ (star-TEM®, Pentapharm, Munich, Germany) and recordingswere allowed to proceed for at least 120 min. The final concentration ofrTF in the assay was 44 fM. The thromboelastographic parameters ofclotting time (CT), clot formation time (CFT) and maximum clot firmness(MCF) were recorded in accordance with the manufacturer's instructions.CT is defined as the time from the start of measurement to the start ofclot formation. CFT is defined as the time from the start of clotformation until an amplitude of 20 mm is reached. MCF is the maximumdifference in amplitude between the two traces during the assay.

Addition of full length TFPI to FVIII-inhibited whole blood in theabsence of TFPI inhibitory peptides substantially inhibited coagulationas indicated by a marked increased in clot time (FIGS. 72A-72C). JBT2528and JBT1837 improved global hemostatic parameters of FVIII-inhibitedplasma (EC₅₀=11 nM and 4 nM, respectively). JBT1837 and JBT2548 improvedcoagulation by reducing the clot time to normal levels in aconcentration dependent manner (FIGS. 72B and 72C; open circles). Thesepeptides failed to reach clot times of FVIII-inhibited blood atincreased TFPI concentrations (e.g., 10 nM). In contrast, concentrationsof JBT2547 above 100 nM fully restored normal coagulation ofFVIII-inhibited whole blood to which 2 nM and 10 nM full length TFPI wasadded.

This example further confirms that TFPI-binding peptide complexesefficiently neutralize TFPI and restore normal coagulation even atincreased TFPI concentrations.

Example 23

Platelets contain full length TFPI, which is released to plasma uponplatelet activation and results in a platelet TFPI concentration inplasma that approximates 50-75% of the full length TFPI in plasma atsites of injury. This example demonstrates inhibition of platelet TFPIby peptides of the invention.

Blood Collection and Plasma Preparation:

Blood samples were collected from a number of different donors. Ninevolumes of venous blood was drawn by venipuncture into 1 volume of 1.09tri-sodium citrate with or without 500 μg/ml CTI and centrifuged at250×g for 15 minutes for the preparation of platelet-rich plasma (PRP)or at 2860×g for the preparation of platelet-poor plasma (PPP). PPP wasaliquoted, snap-frozen and stored at −80° C. until use. In experimentsin which a normal plasma pool (NP) was used, blood collected from morethan 25 healthy individuals was centrifuged at 2000×g for 15 minutes toseparate plasma from blood cells, and again at 11000×g for 5 min toobtain platelet-poor plasma.

Platelet Isolation:

Blood (45 ml) was collected on 7.5 ml acid citrate/dextrose (ACD, 80 mMtrisodium citrate, 52 mM citric acid, 183 mM glucose) and centrifugedduring 15 min at 248×g. To remove residual erythrocytes, the supernatant(platelet rich plasma, PRP) was centrifuged for an additional 5 minutesat 248×g. The PRP was subsequently centrifuged during 15 minutes at 2760rpm (1360×g) to spin down the platelets. The platelet pellet was washedtwice by resuspending in 20 ml (first wash) and 15 ml (second wash)platelet buffer (10 mM HEPES, 136 mM NaCl, 2.7 mM KCl, 2.0 mM MgCl₂,0.5% bovine serum albumin and 0.2% glucose, pH 6.6), followed bycentrifugation during 15 minutes at 2760 rpm (1360×g). After the secondwash step, the platelets were resuspended in 3.5 ml platelet buffer (pH7.5) and the platelet concentration was determined in a Coulter counter.Finally, the platelet suspension was diluted to the required plateletconcentration by dilution with platelet buffer (pH 7.5) and 40 μl of a25 mg/ml solution of the synthetic peptide Arg-Gly-Asp-Ser (RGDS) wasadded per 3.5 ml platelet suspension to inhibit platelet aggregationduring storage at room temperature.

Functional Characterization of Platelet TFPI:

TFPI was released from isolated platelets by activating the platelets at37° C. during 15 min with 100 ng/ml convulxin. The platelets were spundown by centrifugation (15 min at 2800×g) in an Eppendorf centrifugetube, and the supernatant was collected as source of platelet TFPI.Platelet TFPI was quantified with a full length TFPI ELISA usingrecombinant TFPI as standard. The functional activity of platelet TFPIwas compared to the activity of recombinant TFPI in two assaysystems: 1) inhibition of FVIIa-catalysed FX activation in a modelsystem and 2) inhibition of thrombin generation in TFPI-deficientplasma.

In the model system assay, the effect of varying concentrationsrecombinant or platelet TFPI on TF-FVIIa catalysed FX activation wasdetermined at 37° C. in a 25 mM HEPES (pH 7.7), 175 mM NaCl, 5 mg/mlbovine serum albumin (BSA) buffer containing 2 pM FVIIa, 5 nM TF, 100 nMFX, and 400 uM Fluophen FXa. In this assay FVIIa, TF, TFPI and FluophenFXa were preincubated for 7 minutes at 37° C. in the HEPES buffer and FXactivation was initiated by adding FX. Progress curves of fluophen FXaconversion by the FXa generated were determined in Fluoroskan Ascent®reader (Thermo Labsystems, Helsinki, Finland) and corrected forconsumption of Fluophen FXa and so-called inner filter effects(Udenfriend S, Fluorescence Assay in Biology and Medicine. New York,Academic Press, 1996, vol 1, pp 13, 118, vol 2, pp 182-185) by themethodology that is also used for the correction of thrombin generationcurves for substrate consumption (Hemker et al., Pathophysiol HaemostThromb, 32, 249-53 (2002)). Time courses of FXa generation and theeffects of TFPI thereon were obtained by taking the first derivative ofthe corrected progress curves of Fluophen FXa conversion. Alternatively,the effect of TFPI on TF-FVIIa-catalysed FX activation was also followedwith a FXa-specific chromogenic substrate, e.g., 125 μM CS-11(65).

In the thrombin generation assay, TFPI-depleted plasma was reconstitutedwith varying amounts of recombinant TFPI or platelet-derived TFPI, andthrombin generation was determined using the Calibrated AutomatedThrombogram (CAT) method described by Hemker et al., supra. Thrombingeneration was initiated in plasma by addition of varying concentrationsof recombinant TF (0.1-10 pM), 16 mM CaCl₂, 30 μM phospholipid vesicles(1,2-dioleoyl-sn-glycero-3-phosphoserine/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine/1,2-dioleoyl-sn-glycero-3-phosphatidylcholine,20/20/60, M/M/M) and 30-50 μg/ml corn trypsin inhibitor (CTI). Inexperiments in PRP, no phospholipid vesicles were added to the plasma.The thrombin activity in plasma was monitored continuously with thefluorogenic substrate Z-Gly-Gly-Arg-AMC (BACHEM, Bubendorf,Switzerland). Fluorescence was read in a Fluoroskan Ascent® reader(Thermo Labsystems, Helsinki, Finland) and thrombin generation curveswere calculated using Thrombinoscope™ software (Thrombinoscope,Maastricht, The Netherlands).

Analysis of the Effect of Peptides of the Invention on Recombinant-,Plasma-, and platelet TFPI:

The effects of TFPI binding peptides on the anticoagulant activity ofTFPI was tested in model systems (inhibition of FXa andTF-FVIIa-catalysed FX activation by TFPI) and in plasma (NP, PPP, PRP orTFPI-depleted plasma reconstituted with recombinant- or platelet-TFPI orplatelets). The effect of peptides on the inhibition of FXa by TFPI wasfollowed in a HNBSA buffer (50 mM HEPES pH 7.7, 175 mM NaCl, 5 mg/mLBSA) containing 125 μM CS-11(65), 1 mM EDTA or 3 mM CaCl₂ and, ifpresent, varying concentrations of peptide and TFPI, 80 nM protein Sand/or 30 μM phospholipid vesicles (20:60:20 DOPS/DOPC/DOPE), which waspreincubated for 10 minutes at 37° C. hFXa was added and the increase inabsorbance at 405 nm was followed in an Ultra Microplate Reader(Bio-Tek, Burlington, Vt., USA) until a steady state rate of chromogenicsubstrate conversion was achieved (˜60 min). Progress curves ofchromogenic substrate conversion were fitted to the integrated rateequation for slow-tight binding inhibition (Huang et al., J Biol Chem,268, 26950-55 (1993)):A _(t) =A ₀+(v _(s) ·t)+(v ₀ −v _(s))·(1−exp(−k _(obs) ·t))/k _(obs)in which A_(t) is absorbance at 405 nm at time t; A₀ is initialabsorbance at 405 nm; v_(s) is final steady-state velocity; v₀ isinitial velocity; k_(obs) is apparent rate constant for the transitionfrom v₀ to v_(s) (FXa-TFPI to FXa-TFPI*). The values v₀ and v_(s)relative to rates of chromogenic substrate conversion by FXa in theabsence of TFPI, represent the extent of loose and tight FXa-TFPIcomplex formation.

The effect of peptides on the inhibition of TF-FVIIa-catalysed FXactivation by TFPI was determined at 37° C. in a 25 mM HEPES (pH 7.7),175 mM NaCl, 5 mg/ml bovine serum albumin (BSA) buffer containing 2 pMFVIIa, 5 nM TF, 100 nM FX, 400 μM Fluophen FXa and varying amounts ofpeptide and TFPI (recombinant- or platelet-derived). In this assayFVIIa, TF, TFPI, peptide, and Fluophen FXa were preincubated for 7minutes at 37° C. in the HEPES buffer, and FX activation was initiatedby adding FX. Progress curves of Fluophen FXa conversion by the FXagenerated were determined in Fluoroskan Ascent® reader (ThermoLabsystems, Helsinki, Finland) and corrected for consumption of FluophenFXa and so-called inner filter effects by the methodology that is alsoused for the correction of thrombin generation curves for substrate. Thetime courses of FXa generation and the effects of TFPI thereon wereobtained by taking the first derivative of the corrected progress curvesof Fluophen FXa conversion. Alternatively, the effect of TFPI onTF-FVIIa-catalysed FX activation was also followed with a FXa-specificchromogenic substrate e.g. 125 μM CS-11(65).

The effect of peptides on plasma- and platelet TFPI was assessed bymeasuring their effects on thrombin generation determined as describedbelow in NP, PPP, PRP or in TFPI-depleted plasma reconstituted withrecombinant- or platelet-TFPI or platelets. In experiments in PRP or inTFPI-depleted plasma reconstituted with platelets, the platelets wereeither not-activated or activated with 80 ng/ml convulxin or 40 ug/mlHorm collagen (final concentrations in the well). To simulatehaemophilia plasma, thrombin generation experiments were performed inthe presence of a goat inhibitor plasma of which 1 μl was added to 80 μlplasma to neutralize FVIII.

In thrombin generation experiments, the plasma was added to themicrotiter plate and, if present, mixed with proper amounts ofphospholipids, platelets, TFPI, anti-FVIII, anti-TFPI antibodies,peptide and incubated for 7 minutes at 37° C. After this preincubation,TF and platelets activator (if present) were added immediately, followedby a mixture fluorogenic substrate/CaCl₂ mixture (FluCa) which initiatesthrombin generation.

Results:

The platelet supernatant TFPI inhibited TF-FVIIa-catalysed FXinactivation in a concentration-dependent manner. The inhibition byplatelet supernatant was prevented by a cocktail of anti-TFPIantibodies, which underscores that it was TFPI in the plateletsupernatant that is the inhibitor of TF-FVIIa-catalysed FX activation.However, rates of FX activation in the presence of platelet supernatantand anti-TFPI were higher than the rate of FX activation withoutplatelet supernatant, suggesting that the platelet supernatant containeda small amount of a FX activator, e.g., FVIIa. This suggests that thedata is an underestimation of the platelet TFPI concentration due to thefact that more FVIIa has to be inhibited when platelet supernatant ispresent in the assay mixture than in the assay mixture in which thecalibration curve is made with recombinant TFPI in buffer.

The functional activity of platelet TFPI was also tested inTFPI-deficient plasma and compared to the activity of the recombinantnon-glycosylated TFPI. The concentrations of platelet TFPI fromdifferent donors were determined with an ELISA in which the recombinantnon-glycosylated TFPI was used as calibrator. Although no titrationswere made with the different TFPI preparations in TFPI-deficient plasma,inspection of the effects of platelet- and recombinant TFPI on thrombingeneration showed that, on a concentration basis, platelet andrecombinant TFPI exhibit similar anticoagulant activities.

JBT1837, JBT1857, JBT1837+JBT1857, and JBT2547 (50 nM) blocked theability of platelet TFPI to inhibit TF-FVIIa-catalysed FX activation.The fusion peptide was most effective and completely blocked theanticoagulant activity of TFPI. JBT1857 was the least effective followedby JBT1837 and the mixture of JBT1837 and JBT1857. Titration withJBT2547 demonstrated that a concentration of 10 nM almost completelyinhibited the activity of platelet TFPI.

An additional study was performed to further elucidate the effects ofplasma- and platelet-TFPI on the inhibition of thrombin generation inplatelet-rich plasma (PRP) and platelet-poor plasma (PPP). Todiscriminate between the down-regulation of thrombin generation byplatelet TFPI and plasma-derived TFPI, experiments were performed inTFPI-depleted plasma supplemented with platelets (source of plateletTFPI) and/or varying amounts of added purified TFPI (simulatingvariation of plasma TFPI levels). A preliminary experiment inTFPI-depleted plasma supplemented with convulxin-activated plateletsshowed that anti-TFPI antibodies enhanced thrombin generation, whichindicated that TFPI released from the platelets inhibits thrombingeneration.

It appeared that thrombin generation in PRP required TF. In PRP withoutCTI and in PRP to which CTI was added later, the ETP and thrombin peakheight were hardly affected by the TF concentration (TF was notrequired) and only the lag time was shortened at increasing TF. When PRPwas prepared from blood that was collected in citrate plus CTI, both thelag time and the thrombin peak were affected by TF concentration,indicating that experiments performed in PRP require that blood has tobe collected in CTI.

In the experiments described above, the plasma was pre-activated withconvulxin to promote the release of platelet TFPI before initiatingthrombin generation with TF plus Ca²⁺. Since convulxin is not aphysiological trigger, a comparison of thrombin generation in PRP withnon-activated platelets and platelets pre-activated with convulxin,collagen and Ca-ionophore was performed. It appeared that thethrombin-generating capacity of the platelets increased in the order noactivator=ionophore<collagen<convulxin.

Additionally, in the experiments described above, the platelets werepreincubated with activator (pre-activated) for 7 minutes in thepresence of TF, and thrombin generation was initiated with a fluorogenicsub strate/Ca²⁺ mixture. When the platelet activator convulxin is addedto the PRP 10-15 seconds before initiating thrombin generation with thefluorogenic substrate/Ca²⁺ mixture, substantially less thrombin isgenerated than in PRP in which the platelets were pre-activated withconvulxin before thrombin generation was started. In view of theseresults, future studies will not include platelet pre-activation, andcollagen or no activator will be primarily used in experiments in PRP.

The effects of TFPI antagonists were initially tested in PRP preparedfrom blood collected on CTI. Thrombin generation triggered in PRP with0.1 pM TF+collagen was substantially enhanced by both an anti-TFPIcocktail and JBT2547. This shows that TFPI is a major regulator ofthrombin generation on platelets, and that JBT2547 is as effective as ananti-TFPI cocktail in neutralizing the anticoagulant activity of TFPI.TFPI hardly down-regulated thrombin generation in PRP triggered with ahigh amount (10 pM) of TF.

The major part of the thrombin generated in PRP at low TF (1 pM)appeared be formed via the intrinsic Xase and, thus, requires FVIII.Both JBT2547 and an anti-TFPI cocktail substantially increased thrombingeneration in PRP in which FVIII was neutralized with anti-FVIII, acondition representative for hemophilia plasma. JBT2547 also enhancedthrombin generation in TFPI-depleted plasma supplemented with plateletsthat were not activated or that were activated with collagen.

The experiments with JBT2547 were carried out with 4 μM peptide, which,considering the affinity of JBT2547 for TFPI (sub-nM range), likely is alarge excess. Indeed, both in PPP and in PRP, 5-10 nM JBT2547 wassufficient to fully inhibit TFPI.

To test whether TFPI in plasma, either plasma- or platelet-derived, isfully inhibited by the TFPI antibody cocktail or JBT2547, a TFPItitration was performed in PPP and in PRP in the absence and presence ofa TFPI antibody cocktail. Increasing TFPI concentrations on top of theTFPI that is present in plasma plus what is released from plateletsgradually inhibited thrombin generation. In the presence of an TFPIantibody cocktail, all thrombin generation curves were the same,indicating that the TFPI antibody cocktail is not a partial TFPIinhibitor but fully neutralizes TFPI.

In PPP, low concentrations of TFPI added (0.05-0.5 nM) substantiallyinhibited thrombin generation and plots of thrombin generationparameters (log lag time or ln ETP) as a function of total TFPI (plasmaTFPI+added TFPI) could be fitted to a straight line assuming that plasmacontained ˜0.3 nM TFPI. This indicated that the anticoagulant activityof added TFPI is similar to that of the TFPI that is present in plasma.In PRP, much larger amounts of added TFPI were required to inhibitthrombin generation.

The peptide complex JBT2547 enhanced thrombin generation 3-4-fold in PPPwith an effective concentration (EC₅₀) of 2 nM and enhanced thrombingeneration 2-fold in PRP with an EC₅₀ of 8 nM. Thrombin generation inPRP to which an inhibitory FVIII antibody was added to simulate PRP of ahemophilia patient was enhanced 3-fold by the peptide complex. The datadescribed herein establish that peptides of the invention bindplasma-derived, recombinant, and platelet-derived TFPI, and enhancethrombin generation by blocking the anticoagulant effects of plateletand plasma-TFPI.

Example 24

This example describes the hemostatic effect of peptides of theinvention in a murine model of hemophilic joint bleeding.

The goal of the study was to evaluate the effect of an exemplaryPEGylated peptide of the invention on puncture-induced hemarthrosis in amurine model of hemophilia A. Peptide JBT2329 was administered at a doseof 1 mg/kg to FVIII-deficient mice with severe hemophilia (E16 FVIII B6;129S4-F8^(tm 1 kaz)/J) via intravenous tail vein injection. Variousdoses of recombinant FVIII (ADVATE; 10, 50, and 100 IU/kg) also wereadministered alone or in combination with JBT2329. After each productadministration, the right knee joint capsule was punctured with a 30gauge needle to induce hemorrhage. Animals were sacrificed three daysfollowing injury, and bleeding was assessed via gross and histologicalmethods. The animal model and methods of evaluating bleeding are furtherdescribed in Hakobyan et al., Haemophilia, 14, 804-809 (2008). Summarybleeding scores (SBS), which include visual and histological bleedingscores, were assigned to each joint. Administration of a PEGylatedpeptide of the invention prior to injury significantly reduced jointbleeding. The protective effect of 1 mg/kg of JBT2329 was significantlygreater than the effect of 10 IU/kg ADVATE, but less than 100 IU/kgADVATE, as determined by SBS. There was no significant difference in theprotective effect conferred by 1 mg/kg JBT2329 and 50 IU/kg ADVATE. Theresults described herein further confirm that peptides of the inventionprovide a prophylactic or protective effect against bleeding in an invivo model of hemophilia A.

Example 25

This example describes the impact of peptides of the invention on TFPI'sability to bind receptors that mediate clearance and cellulardegradation. The pharmacokinetics of peptide-bound TFPI also isdescribed.

The binding full length TFPI (fl-TFPI) to low density lipoproteinreceptor-related protein (LRP) was studied using a BIAcore T200™ surfaceplasmon resonance assay (GE Healthcare, Chalfont St. Giles, UK) at 37°C. LRP was biotinylated using a biotinylation kit according to themanufacturer's protocol (Thermo Scientific). Following Neutravidinimmobilization (2500 RU) to a Series S Sensor chip C1 (GE Healthcare)using standard amine coupling chemistry according to manufacturer'sprotocols, biotinylated recombinant human LRP-1 Cluster II Fc Chimeraprotein (R&D Systems) was bound to the surface via biotin-NeutrAvidininteractions resulting in 450 RUs. Following immobilization, fl-TFPI wasinjected at a flow rate of 30 μL/min in a single concentration of 10 nMdiluted in running buffer (HBS-N, 0.1% P80, 5 mM CaCl₂). fl-TFPI wassubsequently dissociated by changing the flow to running bufferconditions. When interaction of fl-TFPI and LRP was studied in thepresence of peptides (JBT1837, JBT1857, JBT2329 (JBT1857+40 kD PEG), orJBT2547 (JBT1837+JBT1857)) or polyethylene glycol (40 kD), 1 μM finalconcentration of the peptide or PEG was added to fl-TFPI.

TFPI interacted efficiently with immobilized LRP with fast on- andoff-rates. Truncated TFPI lacking kunitz domain 3 (KD3) and theC-terminus does not interact with LRP. By visual inspection, it wasapparent that fl-TFPI bound to JBT1837, JBT1857 or JBT2329 stillinteracts with LRP. TFPI complexed to the fusion peptide JBT2547resulted in a higher response than that observed for TFPI complexed withsingle peptides. The unchanged association and dissociation kinetics ofthe LRP-fl-TFPI interaction in the presence and absence of a peptidecomplex (JBT2547) suggests that differing results are associated withthe increased molecular weight of the fl-TFPI-JBT2547 complex andpotential simultaneous binding of LRP and peptide to fl-TFPI by JBT2547.A slight decrease in fl-TFPI LRP interaction was observed when TFPI wasbound to JBT2329 (PEGylated JBT1857), whereas a 40 kD PEG alone does notinfluence the assay system, suggesting that a highly hydrated PEG-linkedpeptide slightly interferes with fl-TFPI LRP interaction.

TFPI interaction with asialoglycoprotein receptor (ASGPR) also wasevaluated using a BIAcore 3000™ surface plasmon resonance assay (GEHealthcare, Chalfont St. Giles, UK) at 25° C. Following immobilizationof ASGPR (Novus Biologicals, 470 RU) to a Series S Sensor chip CM5 (GEHealthcare) using standard amine coupling chemistry according tomanufacturer's protocols, fl-TFPI was injected at a flow rate of 30μL/min in the single cycle analysis mode at concentrations ranging from3.84 nM to 150 nM diluted in running buffer (HBS pH 7.4, 0.1% P80, 5 mMCaCl₂). fl-TFPI was subsequently dissociated by changing the flow torunning buffer conditions. When interaction of fl-TFPI and ASGPR wasstudied in the presence of JBT1837, JBT1857, JBT2329, or JBT2547, 1 μMfinal concentration of peptide was added to the fl-TFPI.

Fl-TFPI interacted efficiently with immobilized ASGPR with fast on- andoff-rates. C-terminally truncated TFPI does not interact with ASGPR. Itwas apparent from visual inspection that fl-TFPI bound to JBT1857 orJBT2547 interacted with ASGPR. Like LRP, TFPI complexed to the fusionpeptide JBT2547 generated in a higher response. Also similar to theinteraction of fl-TFPI to LRP, JBT2329 diminished the ASGPR-fl-TFPIinteraction, whereas a 40 kDa PEG alone did not influence the assaysystem. The data suggests that highly hydrated PEG-linked peptides ofthe invention interfere with TFPI-ASGPR interaction via sterichindrance.

To study the impact of TFPI-binding peptides on the pharmacokinetics ofTFPI in vivo, groups of mice (C57B16, male, 20-25 g) were treated witheither human fl-TFPI (775 nM, 5 mL/kg, i.v.), human fl-TFPI complexed toa 10-fold molar excess of JBT2528 (775 nM hu fl-TFPI, 7752 nM JBT2528, 5mL/kg, i.v.) or human fl-TFPI complexed to a 10-fold molar excess ofJBT2534 (775 nM hu fl-TFPI, 7752 nM JBT2534, 5 mL/kg, i.v.). JBT2528 hasthe same peptide sequence as JBT2534, but lacks the 40 kDPEG-modification. As such, JBT2528 serves as a control for a possibleimpact of PEGylation in the assay. At various time points, three micewere sacrificed, blood was taken by heart puncture, and plasma wasisolated and stored frozen (<−60° C.) for further analysis. The samplingtime points were as follows: fl-TFPI, 0.5, 1, 2 minutes;fl-TFPI-JBT2528, 0.5, 1, 2, 3, 5, 8 minutes; and fl-TFPI-JBT2534, 1, 2,5, 10, 20, 35 minutes.

Plasma samples were analyzed for human TFPI with an ELISA which isspecific for human TFPI. For quantification, wells of a microtiter plate(Nunc Maxisorp) were coated with 1 μg/mL of a monoclonal anti-human KD2specific TFPI antibody (Sanquin, White label; MW1845) overnight at 4°C., followed by three wash cycles with TBS containing 0.1% Tween 20(TBST). Wells were blocked for 1 hour at room temperature with TBScontaining 2% non-fat dry milk (BioRad). 100 μL of diluted sample wereapplied to the wells and incubated for 2 hours at room temperature.After washing with TBST (5×), wells were incubated for 1 hour with 0.5μg/mL of a polyclonal rabbit anti hTFPI antibody (ADG72; AmericanDiagnostica), washed 5× with TBST, and incubated further for 1 hour with0.2 μg/mL of a goat anti rabbit HRP labelled antibody (A0545; Sigma).Color was developed by addition of 100 μL of substrate (SureBlue TMP,KLP) and stopping with 50 μL of 1 M HCl. Absorbance at 450 nm wasmeasured with a microtiter plate reader (Thermo Appliskan Reader).Purified endogenous fl-TFPI, expressed by SKHep cells, was used asstandard protein at concentrations of 0.25-16 ng/ml for quantification(Baxter Innovations GmbH). Plasma samples were diluted from 1/20 to1/800 depending on the expected human TFPI concentration.

Human fl-TFPI had a very short half-life and very poor in vivo recovery.At the earliest time point (0.5 min), only one tenth of the expectedTFPI level was observed. The recovery and the half-life of fl-TFPIremained unchanged when fl-TFPI was complexed with the tested peptides.This experiment indicates that PEGylation of a TFPI-binding peptidelikely does not confer a longer half-life to fl-TFPI and does not affectTFPI clearance.

This example demonstrates that representative peptides of the inventiondo not significantly diminish TFPI interaction with clearance receptorsand do not increase the half life of TFPI in vivo.

Example 26

This example demonstrates the ability of peptides of the invention toinhibit proteolytic degradation of TFPI.

TFPI is proteolytically inactivated by several enzymes, includingelastase, thrombin, plasmin, FXa, and chymase. See, e.g., Hamuro et al.,FEBS Journal, 274, 3056-3077 (2007). The impact of fusion proteinJBT2547, as well as the individual peptide subunits JBT1837 and JBT1857,on the proteolytic degradation of fl-TFPI by neutrophil elastase wasstudied. Neutrophil elastase is a representative protease cleaving TFPIwithin Lys86 and Gln90 located between the Kunitz 1 and Kunitz 2domains.

For proteolytic digest of fl-TFPI, 5 nM fl-TFPI were incubated with 5 nMneutrophil elastase (purified from human neutrophils, Calbiochem) in areaction buffer (50 mM Tris, 300 mM NaCl, 10 mM CaCl₂, pH 7.5) at 37° C.The reaction was performed with and without 1 μM JBT1837, JBT1857, orJBT2547. Proteolysis was monitored by Western blot analysis. Aliquotswere taken from the reaction mixture after 0, 5, 15, 30, and 60 minutesand immediately heated at 96° C. for 5 minutes in SDS-loading buffer atreducing conditions. Samples were separated on a 4-20% Tris-glycineSDS-polyacrylamide gel, and the proteins were transferred to a PVDFmembrane. After blocking the membrane with non-fat dry milk, TFPIproteins were detected with a rabbit polyclonal antibody against humanTFPI (AF2974, R&D Systems) and a secondary anti-rabbit-HRP conjugatedantibody (Sigma). SuperSignal West Femto chemiluminescent substrate(Thermo Scientific) was applied to develop a chemiluminescent signalthat was captured on film. The developed bands were quantified bydensitometry.

In the absence of peptides, gradual degradation of fl-TFPI by neutrophilelastase was observed within 1 hour. The ˜43 kD band visualized in theWestern Blot corresponding to intact fl-TFPI almost completelydisappeared, while two cleavage products were formed by cleavage of thepeptide bond T87-T88. When JBT2547 was present, the proteolysis offl-TFPI was blocked during the monitored time, indicating that JBT2547protects fl-TFPI from degradation by elastase in vitro. The individualpeptide subunits mediated different effects on fl-TFPI cleavage. JBT1837protected fl-TFPI from proteolysis for one hour, similar to the resultsobserved for JBT2547. In contrast, JBT1857 had only a minor or no effecton elastase cleavage.

What is claimed:
 1. A peptide complex comprising a first peptide and asecond peptide, wherein (a) the first peptide comprises the structure offormula (XIII)X6001-X6002-X6003-X6004-X6005-X6006-X6007-X6008-X6009-X6010-X6011-X6012-X6013-X6014-X6015-X6016-X6017-X6018-X6019-X6020(XIII) (SEQ ID NO: 3153); wherein X6001 is an amino acid selected fromthe group consisting of F, L, M, Y, 1Ni, Thi, Bta, Dopa, Bhf, C, D, G,H, I, K, N, Nmf, Q, R, T, V, and W; wherein X6002 is an amino acidselected from the group consisting of Q, G, and K; wherein X6003 is anamino acid selected from the group consisting of C, D, E, M, Q, R, S, T,Ede(O), Cmc, A, Aib, Bhs, F, G, H, I, K, L, N, P, V, W and Y; whereinX6004 is an amino acid selected from the group consisting of Aib, E, G,I, K, L, M, P, R, W, Y, A, Bhk, C, D, F, H, k, N, Nmk, Q, S, T and V;wherein X6005 is an amino acid selected from the group consisting of a,A, Aib, C, D, d, E, G, H, K, k, M, N, Nmg, p, Q, R, NpropylG, aze, pip,tic, oic, hyp, nma, Ncg, Abg, Apg, thz, dtc, Bal, F, L, S, T, V, W andY; wherein X6006 is an amino acid selected from the group consisting ofA, C, C(NEM), D, E, G, H, K, M, N, Q, R, S, V, Cit, C(Acm), Nle, I,Ede(O), Cmc, Ed, Eea, Eec, Eef, Nif, Eew, Aib, Btq, F, L, T, W and Y;wherein X6007 is an amino acid selected from the group consisting of I,V, T, Chg, Phg, Tle, A, F, G, K, L, Nmv, P, Q, S, W and Y; wherein X6008is an amino acid selected from the group consisting of F, H, 1Ni, 2Ni,Pmy, Y, and W; wherein X6009 is an amino acid selected from the groupconsisting of Aib, V, Chg, Phg, Abu, Cpg, Tle,L-2-amino-4,4,4-trifluorobutyric acid, A, f, I, K, S, T and V; whereinX6010 is an amino acid selected from the group consisting of A, C, D, d,E, F, H, K, M, N, P, Q, R, S, T, V, W, Y, Nmd, C(NEM), Aib, G, I, L andNmf; wherein X6011 is an amino acid selected from the group consistingof A, a, p, Sar, c, hcy, Aib, C, K, and Nmg; wherein X6012 is an aminoacid selected from the group consisting of Y, Tym, Pty, Dopa and Pmy;wherein X6013 is an amino acid selected from the group consisting ofAib, C, F, 1Ni, Thi, Bta, A, E, G, H, K, L, M, Q, R, W and Y; whereinX6014 is an amino acid selected from the group consisting of A, Aib, C,C(NEM), D, E, K, L, M, N, Q, R, T, V, Hcy, Bhe, F, G, H, I, P, S, W andY; wherein X6015 is an amino acid selected from the group consisting ofR, (omega-methyl)-R, D, E and K; wherein X6016 is an amino acid selectedfrom the group consisting of L, Hcy, Hle and Aml; wherein X6017 is anamino acid selected from the group consisting of A, a, Aib, C, c, Cha,Dab, Eag, Eew, H, Har, Hci, Hle, I, K, L, M, Nle, Nva, Opa, Orn, R, S,Deg, Ebc, Eca, Egz, Aic, Apc, Egt, (omega-methyl)-R, Bhr, Cit, D, Dap,E, F, G, N, Q, T, V, W and Y; wherein X6018 is an amino acid selectedfrom the group consisting of A, Aib, Hcy, hcy, C, c, L, Nle, M, N, R,Bal, D, E, F, G, H, I, K, Q, S, T, V, W and Y; wherein X6019 is an aminoacid selected from the group consisting of K, R, Har, Bhk and V; andwherein X6020 is an amino acid selected from the group consisting of K,L, Hcy, Ami, Aib, Bhl, C, F, G, H, I, Nml, Q, R, S, T, V, W and Y and(b) the second peptide comprises the structure of formula (XIV):X7001-X7002-X7003-X7004-X7005-X7006-[X7007-X7008-X7009-X7010-X7011-X7012-X7013-X7014-X7015-X7016-X7017-X7018]-X7019-X7020-X7021-X7022-X7023(XIV) (SEQ ID NO: 3154), wherein X7001 is either present or absent,whereby in case X7001 is present it is an amino acid selected from thegroup consisting of A, C, C(NEM), D, E, F, G, H, I, K, L, P, R, S, T, Vand W; wherein X7002 is either present or absent, whereby in case X7002is present it is an amino acid selected from the group consisting of A,C, C(NEM), D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y; whereinX7003 is an amino acid selected from the group consisting of A, F, I, K,L, R, S, T, V, W and Y; wherein X7004 is an amino acid selected from thegroup consisting of A, D, E, F, G, I, K, L, R, S, T, V and W; whereinX7005 is R or W; wherein X7006 is an amino acid selected from the groupconsisting of F, H, I, K, L, R, V and W; wherein X7007 is an amino acidselected from the group consisting of Orn, homoK, C, Hcy, Dap and K;wherein X7008 is an amino acid selected from the group consisting of A,G, R, S and T; wherein X7009 is an amino acid selected from the groupconsisting of a, A, I, K, L, M, m, Moo, Nle, p, R, Sem and V; whereinX7010 is an amino acid selected from the group consisting of A, G, I, K,L, P, R, S, T and V; wherein X7011 is an amino acid selected from thegroup consisting of D, E, G, S and T; wherein X7012 is an amino acidselected from the group consisting of A, a, D, d, E, e, F, f, G, I, K,k, L, 1, M, m, Moo, Nle, nle, P, p, R, r, S, s, Sem, T, t, V, v, W andw; wherein X7013 is an amino acid selected from the group consisting ofA, C, C(NEM), Con, Con(Meox), D, d, E, e, Eag, F, G, I, K, L, N, R, S,s, T, V and W; wherein X7014 is an amino acid selected from the groupconsisting of A, D, E, F, G, I, K, L, M, R, S, T, V and W; wherein X7015is an amino acid selected from the group consisting of A, D, E, F, G, I,K, L, M, Nle, R, S, T, V and W; wherein X7016 is an amino acid selectedfrom the group consisting of A, D, E, F, I, K, L, M, Moo, Nle, R, S,Sem, T, V, W and Y; wherein X7017 is an amino acid selected from thegroup consisting of A, D, E, F, G, I, K, L, R, S, T, V, W and Y; whereinX7018 is an amino acid selected from the group consisting of C and D;wherein X7019 is an amino acid selected from the group consisting of A,F, I, L, S, T, V and W; wherein X7020 is an amino acid selected from thegroup consisting of F and W; wherein X7021 is an amino acid selectedfrom the group consisting of I, L and V; wherein X7022 is an amino acidselected from the group consisting of A, D, E, F, G, I, K, L, P, R, S,T, V and W; wherein X7023 is either present or absent, whereby in caseX7023 is present it is an amino acid selected from the group consistingof A, C, C(NEM), Con, Con(Meox), D, E, Eag, F, G, I, K, L, R, S, T, V, Wand Y; wherein the peptide comprises as a cyclic structure generated bya linkage between X7007 and X7018; and wherein the C-terminus of thefirst peptide is linked to the N-terminus of the second peptide via alinker moiety.
 2. The peptide complex of claim 1, wherein wherein X6001is an amino acid selected from the group consisting of 1Ni, Bta, Dopa,F, L, Y and M; wherein X6002 is Q; wherein X6003 is an amino acidselected from the group consisting of D, E, S, M, Q, R, T and C; whereinX6004 is an amino acid selected from the group consisting of K, Aib, L,P, R, E, G, I, Y, M and W; wherein X6005 is an amino acid selected fromthe group consisting of p, Nmg, NpropylG, aze, pip, tic, oic, hyp, a,Aib, D, d, G, H, K, k, N, Q, R, A, E, C and M; wherein X6006 is an aminoacid selected from the group consisting of C, E, K, R, S, V, C(Acm),Nle, C(NEM), I, Cit, A, D, G, H, Q and M; wherein X6007 is an amino acidselected from the group consisting of Tle, V and I; wherein X6008 is anamino acid selected from the group consisting of H, 1Ni, 2Ni, Pmy, F andY; wherein X6009 is V, Abu or Tle; wherein X6010 is an amino acidselected from the group consisting of D, P, C, T, A, E, K, M, N, Q, R,F, H, S, V, W and Y; wherein X6011 is an amino acid selected from thegroup consisting of G, a, c, hcy and Sar; wherein X6012 is Y; whereinX6013 is an amino acid selected from the group consisting of F, 1Ni, Btaand C; wherein X6014 is an amino acid selected from the group consistingof Aib, C, E, Hcy, A, D, K, L, M, N, Q, R, T, V and Aib; wherein X6015is R; wherein X6016 is an amino acid selected from the group consistingof L, Aml, Hle and Hcy; wherein X6017 is an amino acid selected from thegroup consisting of A, Aib, C, c, Aic, Eca, Deg, Cha, Dab, Dap, Eag,Eew, H, Har, Hci, Hle, K, Nle, Nva, Opa, Orn, R, I, L, S and M; whereinX6018 is an amino acid selected from the group consisting of A, Aib, C,c, L, Hcy, N, M and R; wherein X6019 is K; and wherein X6020 is an aminoacid selected from the group consisting of L, Aml, Hcy and K.
 3. Thepeptide complex of claim 1, wherein X7001 is an amino acid selected fromthe group consisting of A, D, F, G, H, K, L and S; wherein X7002 is anamino acid selected from the group consisting of H, F, M and R; whereinX7003 is an amino acid selected from the group consisting of F and Y;wherein X7004 is K; wherein X7005 is W; wherein X7006 is an amino acidselected from the group consisting of F and H; wherein X7007 is C;wherein X7008 is an amino acid selected from the group consisting of A,G and S; wherein X7009 is an amino acid selected from the groupconsisting of M, Sem and V; wherein X7010 is an amino acid selected fromthe group consisting of K, P and R; wherein X7011 is D; wherein X7012 isan amino acid selected from the group consisting of F, L, 1, M and Sem;wherein X7013 is an amino acid selected from the group consisting of D,G, K and S; wherein X7014 is G; wherein X7015 is an amino acid selectedfrom the group consisting of I and T; wherein X7016 is an amino acidselected from the group consisting of D, F, M, Sem and Y; wherein X7017is an amino acid selected from the group consisting of S and T; whereinX7018 is C; wherein X7019 is an amino acid selected from the groupconsisting of A and V; wherein X7020 is W; wherein X7021 is V; whereinX7022 is an amino acid selected from the group consisting of F, L, K, R,P and W; wherein X7023 is either present or absent, whereby in caseX7023 is present it is an amino acid sequence selected from the groupconsisting of A, D, F, M, S and Y; and wherein the peptide comprises acyclic structure generated by a linkage between X7007 and X7018.
 4. Thepeptide complex of claim 1, wherein the linker moiety is about 5-50 Å inlength.
 5. The peptide complex of claim 4, wherein the linker moiety isabout 10-30 Å in length.
 6. The peptide complex of claim 1, wherein thelinker moiety comprises the structure Z₁₋₂₀, wherein Z is an amino acid,hydroxy acid, ethylene glycol, propylene glycol, or a combination of anyof the foregoing.
 7. The peptide complex of claim 6, wherein Z is G, s,S, a, A, Bal, Gaba, Ahx, Ttds, or a combination of any of the foregoing.8. The peptide complex of claim 1, wherein the linker moiety is attachedto the first peptide and/or the second peptide via an oxime, ahydrazide, a succinimide, a thioether, a triazole, a secondary amine, anamide, or a disulfide.
 9. The peptide complex of claim 1, wherein theamino acid sequence of the first peptide is at least 80% identical toSEQ ID NO: 178 or SEQ ID NO:
 4261. 10. The peptide complex of claim 1,wherein the amino acid sequence of the second peptide is at least 80%identical to SEQ ID NO:
 1044. 11. The peptide complex of claim 1comprising the amino acid sequence set forth in SEQ ID NO:
 4260. 12. Apharmaceutical composition comprising the peptide complex of claim 1 anda pharmaceutically acceptable carrier.
 13. A peptide consisting of theamino acid sequence selected from the group consisting of SEQ ID NOs:1337-1355 4240-4241 and 4245-4268.
 14. The peptide complex of claim 1,wherein the linker moiety is about 1-100 Å in length.
 15. The peptidecomplex of claim 2, wherein X7001 is an amino acid selected from thegroup consisting of A, D, F, G, H, K, L and S; wherein X7002 is an aminoacid selected from the group consisting of H, F, Y, M and R; whereinX7003 is an amino acid selected from the group consisting of F and Y;wherein X7004 is K; wherein X7005 is W; wherein X7006 is an amino acidselected from the group consisting of F and H; wherein X7007 is C;wherein X7008 is an amino acid selected from the group consisting of A,G and S; wherein X7009 is an amino acid selected from the groupconsisting of M, Moo, Sem and V; wherein X7010 is an amino acid selectedfrom the group consisting of K, P and R; wherein X7011 is D; whereinX7012 is an amino acid selected from the group consisting of F, L, 1, Mand Sem; wherein X7013 is an amino acid selected from the groupconsisting of D, G, K and S; wherein X7014 is G; wherein X7015 is anamino acid selected from the group consisting of I and T; wherein X7016is an amino acid selected from the group consisting of D, F, M, Sem andY; wherein X7017 is an amino acid selected from the group consisting ofS and T; wherein X7018 is C; wherein X7019 is an amino acid selectedfrom the group consisting of A and V; wherein X7020 is W; wherein X7021is V; wherein X7022 is an amino acid selected from the group consistingof F, L, K, R, P and W; wherein X7023 is either present or absent,whereby in case X7023 is present it is an amino acid sequence selectedfrom the group consisting of A, D, F, M, S and Y; and wherein thepeptide comprises a cyclic structure generated by a linkage betweenX7007 and X7018.
 16. The peptide complex of claim 15, wherein the firstpeptide comprises an amino acid sequence at least 80% identical to SEQID NO: 178 or SEQ ID NO:
 4261. 17. The peptide complex of claim 16,wherein the first peptide comprises SEQ ID NO:
 4261. 18. The peptidecomplex of claim 16, wherein the second peptide comprises an amino acidsequence at least 80% identical to SEQ ID NO:
 1044. 19. The peptidecomplex of claim 17, wherein the second peptide comprises an amino acidsequence at least 80% identical to SEQ ID NO:
 1044. 20. The peptidecomplex of claim 17, wherein the second peptide comprises SEQ ID NO:1334.
 21. A method for treating a subject suffering from a diseasewherein the disease is a blood coagulation disorder or being at risk ofsuffering from a disease, the method comprising administering to thesubject a pharmaceutical composition of claim 12.